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D.P.H.Camb., L.R.C.P., M.R.C.S., L.D.S.Eng. 

Bacteriologist and Lecturer on Bacteriology, National Dental Hospital ; 

Sen. Dental Surgeon, Seamen's Hospitals ; lion. Lecturer on Hygiene 

of the Mouth, London School of Tropical Medicine ; late Demonstrator 

of Practical Dentistry, Guy's Hospital Dental School 






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The micro-organisms of the mouth include species belonging to 
higher orders than the Bacteria or Schizomycetes. I have there- 
fore used the wider term Mycology, in preference to Bacteriology, as 
the title of the present work. 

Bacteriology has developed to such an extent that at the present 
time some fifteen hundred organisms have been described and a large 
proportion of them are to be met with occasionally in the mouth. 
It is impossible to include in the scope of the present work, all the 
bacteria found from time to time in the oral fluids, especially as 
environment, food, dust and other causes determine to a great extent 
the species of the buccal flora ; at the same time a certain number 
of organisms, many of them well known to bacteriologists in other 
situations, are so frequently found that they deserve consideration as 
mouth bacteria ; a few bacteria and some higher forms related to the 
Hyphomycetes are to be found in the mouth only, I have therefore 
included in the following pages those bacteria frequently living in the 
buccal cavity although they are also found in other places, and those 
special organisms so far known in no other region than the mouth. 
An attempt is made for the first time to produce a practical text-book 
dealing with mouth bacteria, and although primarily intended for 
the use of students of dental surgery it is hoped the collection of 
facts related to the Mycology of the Mouth may be of assistance to 
those engaged in research. As the work is mainly written for 
students a good deal of stress is laid upon the practical details of 
laboratory routine, bacteriology requiring more laboratory experi- 
ence perhaps than any other subject. General principles of biology, 
sterilization, &c, are also given at some length, for only with a 
thorough knowledge of general principles is it possible to attempt 
the study of individual organisms and the student is advised to 

viii. Preface 

master general facts before proceeding to minute descriptions of the 
organisms themselves. 

In bacteriological work a definite plan should be followed both 
for the benefit of the student and for the interests of the science at 
large ; recognised methods should always be adopted in the first 
place, and full details of others accurately given, the plan given in 
the text and the suggestions on the " Study of Cultivations," will, 
it is hoped, prove useful guides. 

The pathogenic bacteria of the mouth are given at some length 
and are of great importance in the relation of pathological conditions 
of the mouth to general disease, a relationship long since recognised 
by dental surgeons competent to judge, although the subject has 
only recently received the general attention it deserves. Mouth 
pathology with probable, but so far undescribed bacteriology, is only 
too common and it is hoped that by calling attention to the gaps in 
our knowledge others may be induced to investigate these unde- 
termined problems. 

Several of the organisms given in the latter portion of the book 
may possibly be synonymous, but as so far I have had no opportunity 
of carefully testing all, the descriptions originally given are adhered 
to. The question of immunity is touched upon and a general 
statement of fact given, but it is impossible within the scope of 
a text-book of this description to discuss the matter in full ; my 
intention is rather to give the main points without going deeply into 
the subject. 

Fermentation and Dental Caries are so closely related that 
a good deal of space is devoted to their consideration ; a general 
resume of dental caries from the point of fermentation physiology 
is naturally associated with the description of the bacteria involved 
in what is a special form of metabiotic putrefaction. 

In dealing with the special mouth bacteria references are given 
to the original papers to which the student has the opportunity to 
refer if he so wishes. 

In the appendix are given some practical hints on the choice and 
use of the microscope, and the scheme for a system of describing 
cultivations of bacteria recently suggested by Chester in his 
" Determinative Bacteriology," definite but general terms taking 
the place of lengthy descriptions, the latter often proving inaccurate 
from the variation of individual organisms. The system has much 
to commend it. 

Preface ix. 

In writing this book the various standard text-books have been 
freely consulted, among them may be mentioned : " Sternberg's 
Bacteriology," " Macfarland's Pathogenic Bacteria," " Muir and 
Eitchie's Text-book of Bacteriology," " Miller's Micro-organisms 
of the Human Mouth," and the works of Lefar, Hueppe, Du Barry, 
Pflugge, Migula, Lehman and Neumann, and others. 

In conclusion, I acknowledge with many thanks the loan of 
a number of illustrations, which are duly acknowledged. 

My thanks are also warmly accorded to my friend, Dr. J. W. 
Eyre, for many valuable suggestions and for the performance of 
many inoculation experiments at various times. 

Mr. Cyril Hill has also kindly assisted in photographing 
apparatus and in the matter of proofs. 


Bacteriological Laboratory, 

National Dental Hospital. London. 
November, 1902. 



I. Introduction. — Classification. — Morphology . . . . . . 1 

II. Biology 14 

III. Sterilization . . . . . . . . . . . . . . 29 

IV. Methods of Observing Bacteria— Microscopical Methods 40 
V. Methods of Observing Bacteria -Methods of Cultivation 51 

VI. Susceptibility and Immunity . . . . . . . . . . 71 

VII. Pathogenic Bacteria of the Mouth . . . . . . . . 81 

Streptococcus Pyogenes Bacillus Friedlander 

Staphylococcus Aureus Bacillus Influenza? 

Staphylococcus Albus Bacillus Pyocyaneus 

Staphylococcus Citreus Streptothrix Actinomyces 

Diplococcus Pneumonia 1 Bacillus Pulp* Pyogenes 

Micrococcus Tetragenous Bacillus Gingiva? Pyogenes 

Bacillus Diphtheria* Micrococcus Gingiva? Pyogenes 

Bacillus Tuberculosis Bacillus Dentalis Viridens 

VIII. Bacteria in Dental Caries . . . . . . . . . . . . 133 

Streptococcus Brevis Bacillus Mesentericus Ruber 

Bacillus Necrodentali^ Bacillus Mesentericus Fuscus 

Sarcina Lutea Bacillus Mesentericus Furvus 

Sarcina Aurantiaca Bacillus Liquefasciens Fluorescens 

Bacillus Gangra?na? Pulp* Bacillus Subtilis 

Bacillus Mesentericus Vul- Proteus Zenkeri 

gatus Bacillus Plexiformis 

IX. Bacteria in Tooth Pulps . . . . . . . . . . . . 1GG 

X. Bacteria in Dento-Alveolar Abscesses . . .. ..• .. 170 

Staphylococcus Viscosus 

XI. Bacteria in Pyorrhcea Alveolaris . . . . . . . . 175 

XII. Bacteria only known to occur in the Mouth . . . . . . 181 

Spirillum Sputugenum Bacillus Maximus Buccalis 

Spirochete Dentium Streptotbrix Buccalis 

Leptothrix Racemosa Leptothrix Innominata 

Leptothrix Placoides Alba Iodococcus Vaginatus 

XIII. Saprophytic Bacteria of Mouth not described in Previous 

Sections . . . . . . . . . . . . . . . . 206 

Bacillus Coli Commune Bacillus " B." (Vignal) 

Bacillus Luteus Bacillus " F." (Vignal) 

Bacillus Buccalis Minutis V. Finkler-Prior 
Bacillus Fortuitus Micrococcus Roseus 

Appendix . . . . . . . . . . . . . . . . 215 


1. — Morphological forms of bacteria ... ... ... ... 5 

2. — Types of spore formation. Types of spore germination. Types 

of flagellation ... ... ... ... ... ... 10 

3.— Involution forms of bacteria ... ... ... ... 11 

4. — Hot air sterilizer ... ... ... ... ... ... 31 

5. — Copper box for sterilizing petri dishes ... ... ... 31 

6.— Petri dish ... ... ... ... ... ... 32 

7. — Glass capsule ... ... ... ... ... ... 32 

8. — Koch's steam sterilizer ... ... ... ... ... 33 

9. — Autoclave ... ... ... ... ... ... 34 

10. — Koch's blood serum inspissator ... ... ... ... 35 

11. — tnglazed porcelain filters (Pasteur Chamberland) ... ... 36 

12. — Coverglass jar ... ... ... ... ... ... 40 

13. — Hanging drop slide ... ... ... ... ... 41 

14. — Stand to hold bottles of stains ... ... ... ... 45 

15. — Boston's coverslip forceps ... ... .. ... ... 48 

16. — Cornet's coverslip forceps . . ... ... ... ... 48 

17. — Bacillus typhi abdominalis, showing flagella ... ... ... 50 

18. — Apparatus for filling tubes with nutrient solutions ... ... 52 

19. — Erlenmeyer flask ... ... ... ... ... ... 53 

20. — Hot water funnel ... . . ... ... ... ... 55 

21. — Potato cutter ... ... ... ... ... ... 57 

22. — Roux's potato tube ... ... ... ... ... 57 

23. — Platino-iridium inoculating wires ... ... ... ... 60 

24. — Method of inoculating tubes ... ... ... ... 60 

25. — Hearson's biological incubator ... ... ... ... 61 

26. — Hearson's gas valve ... ... ... ... ... 62 

27. — Buchner's tube for anaerobic cultivations ... ... ... 64 

28. — Bullock's anaerobic apparatus ... ... ... ... 65 

29.— Wolff bottle ... ... ... ... ... ... 65 

30. — Filter flask with Pasteur- Chamberland filter ready for filtering 

toxine ... ... ... ... ... ... 68 

31.— Widal Blood Pipette ... ... ... ... ... 76 

xiv. List of Illustrations 


32. — Streptococcus pyogenes in blood, x 1000 ... ... ... 83 

33. — Streptococcus pyogenes, twenty-four hours' agar culture x 1000 83 

34. — Streptococcus pyogenes, twenty-four hours' agar cultivation ... 84 

35. — Streptococcus pyogenes, agar culture, x 1000 ... ... 87 

36. — Streptococcus brevis on epithelial cell direct from mouth, x 1000 88 

37. — Streptococcus brevis, twenty-four hours' agar culture. x 1000 88 

38. — Pipette for collecting pus ... ... ... ... . . .• 97 

39. — Diphtheria bacillus, x 1000 Gram, twenty-four hours' old agar 105 
40. — Diphtheria bacillus, forty-eight hours' old blood serum. Gram. 

x 1000 ... ... ... ... ; ... ... 105 

41. — Bacillus diphtheria cultivation. (Curtis' " Essentials of Practical 

Bacteriology ") ... ... ... ... ... 108 

42. — Bacillus tuberculosis, glycerine agar cultivation (Curtis' " Essen- 
tials of Practical Bacteriology ")... ... ... ... Ill 

43. — Bacillus Friedlander gelatin stab (Curtis' "Essentials of Prac- 
tical Bacteriology ") ... ... ... ... ... 118 

44. — Bacillus pyocyaneus, twenty-four hours' agar cultivation. 

xlOOO ... ... ... ... ... ... 122 

45. — Streptothrix actinoinyces cultivation on glycerine agar (Curtis' 

" Essentials of Practical Bacteriology ") ... ... ... 126 

46. — Dental caries affecting enamel ... ... ... ... 135 

47. — Dental caries affecting dentine ... ... ... ... 144 

48. — Streptococcus brevis, agar culture at twenty-four hours, x 1000 149 

49. — Streptococcus brevis, broth culture twenty-four hours, x 1000 149 
50. — Bacillus mesentericus vulgatus, twenty-four hours' agar culture. 

xlOOO ... ... ... ... ... ... 153 

51. — Bacillus subtilis showing spore formation ... ... ... 158 

52. — Bacillus necrodentalis, forty-eight hours' agar, x 1000 ... 162 

53. — Bacillus plexiformis, gelatin culture, forty-eight hours, x 1000 ... 164 
54. — Bacillus plexiformis, twenty-four hours, decalcified dentine. 

x 1000 ... ... ... ... ... ... 164 

55. — Yeasts, from Eyre's " Bacteriological Technique " ... ... 172 

56. — Various forms of mouth bacteria ... ... ... ... 182 

57. — Leptothrix racemosa, balsam mount (Dr. Leon Williams), x 1000 186 
58. — Leptothrix racemosa, glycerine mount (Dr. Leon Williams). 

x 1000 ... ... ... ... ... ... 187 

59. — Leptothrix racemosa, " fruitful heads " (Dr. Leon Williams). 

x2000 ... ... ... ... ... ... 188 

60. — Leptothrix racemosa, mouth direct, x 1000 ... ... ... 189 

61. — Bacillus buccalis maximus, twenty-four hours' agar, x 1000 ... 191 

62. — Spirillum sputugenum (spirilla forms), mouth direct, x 1000 ... 195 

63. — Spirillum sputugenum (comma forms), mouth direct. x 1000 ... 195 
64. — Spirillum sputugenum freshly isolated, twenty-four hours' agar. 

x 1000 ... ... ... ... ... ... 197 

List of Illustrations 

65. — Spirillum sputugenuru (comma forms), twenty-four hours' agar. 

xlOOO ... ... ... ... 198 

66. — Spirillum sputugenum (spirilla forms), seven days' broth, x 1000 199 

67. — Streptothrix buccalis, mouth direct, x 1000 ... ... 202 

6S. — Streptothrix buccalis, forty-eight hours' agar culture, x 600 ... 202 

69. — Streptothrix buccalis, seven days' potato culture, x 1000 ... 203 

70. — Streptothrix buccalis, five days' agar culture ... ... ... 204 

71. — Bacillus coli commune, twenty-four hours' agar, x 1000 ... 207 

72. — Vibrio Finkler-Prior, twenty-four hours' broth, x 1000 ... 212 

73. — Compound bacteriological microscope (Watson's) ... ... 215 

74. — Fine adjustment of microscope (Watson's) ... ... ... 217 

75. — Characters of gelatin stab cultivations (Eyre, after Chester) ... 219 

76. — Characters of gelatin stab cultivations (Eyre, after Chester) ... 220 

77. — Types of colonies (Eyre, after Chester) ... ... ... 221 

78. — Types of colonies (Eyre, after Chester) ... ... ... 221 

79. — Detailed character of surface of stab cultures (Eyre, after Chester) 222 

80. — Structure of colonies (Eyre, after Chester ... ... ... 222 

81. — Structure of colonies (Eyre, after Chester) ... ... ... 223 

82.— Edge of colonies (Eyre, after Chester) ... ... ... 224 



Bacteria are mhmteuaicell ular organisms forming the lowe st 
groii^jiL-theJii^xtngains, oj^ilojx^riej5s_j3]£n^ 

media t e link betw een Ihe-ammal- and vegetable kingdom of living 
thin gs, related on the one hancLto the llycetozoa, or animal fungi, 
on the other to the Algae . They are divisible into two groups, a 
higher and a lower. The lower, known as Schizomycetes, or fission - 
fungi, are most numerous, comprising the greater number of the 
organisms with which pathological mycology deals. They are all 
microscopic in size, and are rarely more than ^5^00 m - * n one direc- 
tion. To facilitate general description mycologists have adopted a 
standard of measurement designated micron = ioW P ar ^ °^ millimetre, 
or 25IT00 P ar ^ °^ i ncn > which is written ^, the dimensions of an 
organism being expressed as multiples or fractions, e.g., " 2*5 ^ long, 
0-75 iul wide." The lower group of bacteria consists of the relatively 
monomorphous varieties, which are classified according to their 
shape : (a) small globular bodies, occurring singly or associated with 
others, designated cocci ; (b) rod-shaped forms known as bacilli ; (c) 
spiral or corkscrew forms, and the curved fragments of the same, 
called spirilla. The term bacterium is properly applied to micro- 
organisms of the schizomycetal group generally, and is used as such 
in the present work. The higher bacteria are relatively pleo- 
morphous, and may exhibit real or pseudo-branching as in Cladothrix 
dichotoma; their method of reproduction as well as their form is 
allied to the moulds, whilst some of the stages in their life cycle are 


indistinguishable morphologically from the lower group of fission - 

The bacteria as a group are most active chemical agents, split- 
ting up effete animal and vegetable matter into bodies assimilable 
by plants, fixing free nitrogen as on the roots of Leguminosae, and 
assisting in the disintegration of the hardest rocks. A limited 
number produce disease in both animals and plants, and finally van 
Tieghem claims to have demonstrated the presence of the bodies 
of bacteria in coal, where their activity was concerned in the 
rotting of the old coal forests. 

Classification. — Bacteria belong to the vegetable kingdom, and 
are placed under the sub-group of Thallophytes, one of the divisions 
of the Cryptogams, thus : — 

I. Thallophytes. — Simple plants, ( M . _ . T ^ . , , . , 

without leaves, stems, roots, £ ^ ' ' ? chlorophyl. 

or vascular bundles. 1 (2 > A1 S ffi ' ' Containin § chlorophyl. 

II. Bryophyta. — Mosses,with leaves / ... TT , . _ . 

and stems, devoid of true * Hepatin* .. Liverworts. 

roots and vaseular bundles. 1 (4) Musci ' ' Feather mOMeB - 

III. Pteridophyta. — Vascular cryp- ( ,„, _. . ,. TT , .. 

, ■•-,-, . .(.5) Equisetinse Horse-tails, 

togams, with leaves, stems, \ )' T u ,. T ... 

, , , . 4 (6) Lycopodmee Lycopodmm. 

true roots, and vascular j )_; ^. t _r r 

bundles. (t 7 > mwm - •• Ferns - 

The fungi may be divided into two main groups according to 
their mode of growth. 


Schizomycetes . . Fission-fungi. 

Eumycetes . . . c Higher fungi, generally branching. 

The above scheme gives the general position of the bacteria, but 
it is extremely difficult to properly classify all the organisms gener- 
ally included in the term "bacteria" ; more particularly is this the 
case with those species — of which Actinomyces may be taken as an 
example — which are related to the true Schizomycetes on the one 
hand and to the Eumycetes on the other. 

Most of the forms with which bacteriology has to do belong 
however to the Schizomycetes, and it is necessary for convenience 
of description to adopt a classification. 

The grouping generally adopted is Baumgarten's, a modification 
of that first suggested by Cohn, based upon morphological form, 
and although not entirely scientific is at present the most con- 


Baumgarten's classification is as follows : — 

I. — Cocci. \ 

II. — Bacilli. - Species relatively monomorphous. 

III.— Spirilla. j 

I. — Spirulina (Hueppe). \ 

II.— Leptotrichere (Zoph). [ Species relatively pleomorphous. 

III.— Cladotrichese (Cohn). f 

IV. — Streptothrix. j 

Various other methods of classification have been suggested by different 
observers ; thus Du Bary and Hueppe adopt a classification based on spore 
formation : (1) those bacteria forming endogenous spores ; (2) those forming exo- 
genous or arthrospores. Our knowledge of sporulation is as yet too imperfect 
to adopt such as a method of classification. 

Another source of confusion is the different meaning which authors attach 
to their terminology ; for instance, bacterium is used as a general term 
including bacteria generally ; Hueppe uses it in the limited sense of those 
organisms which do not produce endogenous spores, the term bacilli being 
applied to the spore-forming species. Migula calls motile organisms bacilli, 
non-motile ones bacteria. I have adopted the commonly accepted meaning of 
bacillus as a rod-shaped organism, and the term bacterium or bacteria as a 
general one. 

Considerable confusion also exists regarding the loosely applied term lepto- 
thrix, some meaning thereby a thread or special morphological form which is 
common to a considerable number of species. Zoph, who first used the term, 
applied it to a distinct species of the higher bacteria, and it is in this sense it 
is used in the following pages. 

Chester (" Determinative Bacteriology ") suggests the following classifica- 
tion : — 


I. — Cells unbranched, or showing only false branching as in Cladothrix. 

(a) Cells globular, becoming slightly elongated before division, which takes 
place in one, two, or three dimensions. 


(b) Cells short or long, cylindrical, straight, curved or spiral, without sheath ; 
motile or non-motile ; endospores present or absent. 


(c) Cells surrounded by a sheath and arranged in elongated filaments. 


(d) Cells not surrounded by a sheath, arranged in filaments and motile by 
means of an undulating membrane. 


II. Cells short or long, cylindrical or filaments, clavate, cuneate, or irregular 
in form. Without endospores, but with formation of gonidia-like bodies by 
segmentation of the cells. Without flagella. Division at right angles to the 


rod or filament. Not possessed of sheath, but having true dichotornous 

For grouping of species see Chester ("Determinative Bacteriology," p. 54). 

Lehmann and Neumann suggest the following arrangement : — 

(1) Coccacece. 

(2) BacteriacecB. 

(3) Spirillacece. 

(4) Corynebacterium, Diphtheria bacillus. 

(5) Mycobacterium, acid fast organisms (tubercle bacillus). 

(6) Actinomyces. 

Cladothrix, Crenothrix, and Leptothrix are classed as " Higher fission- 

In both these classifications the genus Bacteriacese are divided into two 

(a) Bacterium— not forming endospores. 

(b) Bacilli— forming endospores. 

For further information see Lehmann and Neumann (p. 119 et seq.), where 
proper principles for nomenclature of bacteria are laid down. 

The Lower Bacteria. — (a) Cocci — round, oval, or elliptical cells 
ranging from 0*5 to 2 ^ in diameter. When not spherical the 
greatest diameter is not greater than twice the lesser. They are not 
possessed of motility but often exhibit considerable Brownian move- 
ment (p. 11) with the exception of a few species (e.g., Micrococcus 
agilis of Cohn, Malta fever coccus). Eeproduction is by binary 
fission, and spore formation (endogenous) is unknown. In some 
cocci, particularly the streptococci, large swollen elements with 
increased refractive power are often to be seen ; these have been 
described by Du Bary as a mode of spornlation under the term 
arthrospores (fig. 1, d.) 

The cocci are arranged into groups accordiug to their mode of 

(1) Streptococci. — Division (binary fission) occurring regularly 
in one plane only, the individual elements remain attached by their 
capsules in the form of chains ; in some species the chains may 
attain great length and be composed of a large number of individual 

(2) Staphylococci. — Division occurring irregularly in one plane 
only, the cocci remaining attached by their capsules in irregular 
clumps and masses, compared to bunches of grapes. 

The number of species in these two groups is very large. The 
formation of arthrospores is well marked (Du Bary). 

(3) Diplococci. — Division in one plane regularly, the cocci 

remaining associated in pairs ; both of the foregoing groups exhibit 
this form, the term being applied to a given species when it occurs 
most commonly in the diplococcal form (Diplococcus pneumoniae). 

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*■■■ * .V \_ 

Fig. 1.— Morphological Forms of Bacteria. 
a, Cocci ; b, diplococci, diplococcus with capsule ; c, staphylococci ; d, strepto- 
cocci ; e, tetracoccus and sarcinee ; /, various types of bacilli ; g, streptobacilli ; 
7i, f spirillurn, comma forms and spirochete ; i, streptothrix branched threads. 

(4) Mekismopedia. — Division in two directions in the same 
plane, the cocci remaining attached in groups of four, or "tetrads," 
as Micrococcus tetragenous. 


(5) Saucing. — Division in three planes, one at right angles to 
the other two, the cocci remaining attached in cubical packets of 
eight, which are often associated in masses. These cocci are 
generally of larger size than the other groups. 

The term micrococcus is applied to all the above species, and is 
also used to designate cocci occurring separately, as mono-cocci ; 
such mono-coccal form is common among the staphylococci. 

(b) Bacilli. — Kod-shaped organisms, the greatest diameter being 
more than twice the lesser ; the cells may be long or short, but, as 
a rule, are not wider than 1*0 /*. Division generally takes place at 
right angles to the long axis. The cells of some species have flagella 
and are motile. Two bacilli may remain united by their capsule, 
forming a diplobacillus, or the individual elements remaining united 
in chains a streptobacillus is formed. These two morphological 
forms are not so constant as the corresponding cocci, and are of 
little use in classification. Bacilli frequently grow out into long 
threads, particularly in fluids, to which form the term " leptothrix " 
is often wrongly applied. Endogenous spore formation occurs in 
many species. The spores may be central or terminal in position ; 
round, oval or fusiform in shape. In the latter case the organism 
containing the spore becomes swollen and spindle-shaped and is 
termed a Clostridium. 

(c) Spirilla. — Curved or comma-shaped rods and spiral filaments. 
Eeproduction by binary fission or simple division. Generally motile, 
movement in direction of long axis and rotation upon same axis. In 
the long spiral threads, also termed spirochete, the motility does 
not always appear to be due to the presence of flagella, the organisms 
possessing contractility. The comma-shaped cells generally possess 
flagella, which may be single or multiple and situated at one or 
both poles. 

The term " vibrio " is often applied as a generic term to this group ; some 
authors however use " vibrio " in a special sense, thus Hueppe calls forms without 
endospores " spirochete," " vibrio " those having endospores. Migula applies the 
term vibrio to those organisms with only one or two polar flagella, spirilla those 
with bunches of flagella, and spirochete those without flagella. Fliigge employs 
" vibrio" to denote forms with but slightly marked undulations. 

The Higher Bacteria. — These organisms show a distinct advance 
on the lower group, but at present our knowledge of them is 
fragmentary and includes only isolated species. Some of the 
species show differentiation of the two extremities ; one end. 

may be specialised for attachment, the other for reproduction — a 
method approaching the sporulation of the Moulds, to which the 
High Bacteria are somewhat closely related. The filaments of 
these bacteria are generally segmented, but special methods are 
required to bring out the divisions. There is often a capsule 
common to the whole thread. 

Spirulina (Hueppe). — The cells are sometimes rod-shaped, some- 
times spiral, and in some media may grow out into long spiral, 
undulatory or straight filaments. The threads break up into cocci- 
like reproductive bodies — " arthrospores." Under this head the 
Proteus group was first described ; they are now generally placed 
with the bacilli. 

Leptothrix (Zoph). — Eod-shaped, spherical and filamentous 
forms, the last showing a difference between base and apex. 
Filaments straight or spiral. Spore formation unknown. 

This definition of leptothrix is the one I have adopted in the 
following pages, bacilli forming filaments or threads are not 
included in it. (See later.) 

Cladothrix. — Spherical, rod-shaped, and filamentous forms, the 
latter show pseudo branching. Eeproduction by arthrospores. 

Streptothrix. — Felted mycelium-like filaments, showing true 
dichotomous branching. Club-shaped thickenings appear at the 
ends of some of the threads. Various forms are produced by 
breaking up of threads simulating cocci, bacilli and spirilla ; from 
these new individuals may be formed. 

Bacteria are also classified according to the environment neces- 
sary to their development, as Saprophytes and Parasites. 

The saprophytes are those whose existence is possible apart 
from a living host, and which obtain the nutriment necessary 
for their growth from dead organic matter, or from simple organic 
salts and water. 

The strict or obligatory parasites are unable to exist apart from 
a living host, in whose tissues they multiply, often producing pro- 
found pathological changes ; some few species may exist in the 
tissues of an animal without any harm arising. 

Such bacteria as are capable of leading a saprophytic existence, 
but when gaining access to the tissues of the body will develop there, 
are termed facultative parasites. 

Of the obligatory parasites the leprosy bacillus affords a good 
example ; all attempts at its culture have failed, and it is unknown 


apart from the disease with which its name is associated. The 
cholera spirillum, typhoid bacillus, and most pathogenic bacteria 
are examples of the facultative parasite, which, besides the pro- 
duction of disease by development in living animal tissues, is enabled 
to exist outside the body as a saprophyte. 

There are many transitional forms between the obligatory para- 
site and the saprophytic bacteria ; many of these have only been 
obtained in pure culture within recent years, as, for instance, the 
influenza bacillus and the gonococcus. An organism living a 
parasitic existence as a general rule does not grow in artificial 
media as well as one which has for some time led a purely 
saprophytic existence. For this reason many bacteria require 
some little time before they develop their " laboratory habit." 
It seems not improbable that the various pathogenic bacteria 
which are to-day associated with disease were at one time simple 
saprophytes, and that some of the simple saprophytes, as we know 
them to-day, may yet attain pathogenic powers. 

Not to admit such a development of pathogenic power entails 
the obscession that pathogenic bacteria were created by design to 
destroy human life, and, moreover, such a refusal places us at 
variance w T ith the Monistic conception of the universe, and the 
orderly operation of the laws of evolution with which all observed 
phenomena accord. 

Morphology has been already referred to as the general basis of 
classification. Morphological form, however, is extremely variable, 
so that only the predominating or average form in any given species 
is taken as representing that species. Thus, for instance, the 
Pneumococcus or Diplococcus pneumoniae occurs generally in the 
form of diplococci, but also constantly presents a monococcal and 
streptococcal form. The streptococcus of the mouth occurs in that 
cavity as a diplococcus almost without exception, whilst in the 
majority of culture media the streptococcal form predominates. 
Under certain circumstances the individual cocci may become so 
much elongated that a form allied to a strepto-bacillus is produced. 
It is for this reason, as well as for the fact that three chief morpho- 
logical forms are common to a great number of families, that 
mycologists have adopted the methods of cultivation in the 
determination of species. 

Chemistry. — The determination of the chemistry of the bacterial 
cell was first undertaken by Nencki and Schaffer, who found that 


the bodies of bacteria were very rich in a nitrogenous substance to 
which the term micro-protein was applied ; with nitric acid this 
body does not give the xanthoproteic reaction. The percentage 
of nitrogen is generally about 14*75. 

The bacterial bodies also contain some 3*5 per cent, of fat ; 
Bullock has recently extracted a considerable quantity of fat from 
tubercle bacilli. Bacteria elaborate within their own protoplasm 
various ferments or enzymes and poisons or toxines ; some of these 
are retained within the bacterial cell and may be obtained from the 
washed bodies of bacteria by appropriate methods. Thus Buchner 
obtained an active body, which fermented sugar with the production 
of alcohol, by expressing the contents of washed and dried yeast. 
The fermentation produced in no way differed from that brought 
about by living yeast, the fluid being demonstrably free from living 
organisms. Quite recently Macfadyen 1 and Kowland prepared a 
principle from the washed bodies of typhoid bacilli by triturating 
them with sterile sand in a special apparatus. The glycerine 
extract obtained when injected into rabbits induced an agglutinative 
power in the serum similar to that developed during an attack of 
typhoid fever in the human subject. Some bacteria give a blue 
colouration with acidulated iodine, due to the presence of granu- 
lose ; a number of these are met with in the mouth at various times. 
The butyric acid-bacilli give this blue reaction with iodine, and 
are grouped geuerically as Graiiulobacteria (Lefar). 

Gram's method of staining (p. 46) also depends on some 
chemical difference in the composition of the given organism, some 
retaining the stain, others are quickly decolourised with the alcohol 

Structure. — The bacterial cell consists of a thin cell wall enclosing 
clear and, as far as is at present known, structureless contents. 

The cell wall, allied but not identical with cellulose, is said to be 
of a radiate, honeycombed appearance, and that in this layer is 
situated the colouring matter of the chromogenic organisms, whilst 
the sulphur granules appearing in the Beggiatoa are contained in 
the internal plasma. 

The plasm also contains at times highly refractile granules 
which are not spores ; these granules are said to be homologous 
with the chromatin granules of higher plants from the avidity with 

1 Cent, fur Bak., xxiv., 1902. 


which they take up colouring matter — such granules are to be seen 
in the diphtheria bacillus. 

By a" delicate process of extraction and subsequent staining 
with haematoxylin the central plasm of the organism has been 
shown by Biitschli to be finely reticulated, and that the plasm 
appears differentiated into a central portion and a parietal layer 
besides the cell wall ; the central mass is therefore a large nucleus, 
the parietal layer corresponding to the cytoplasm of higher plants. 


§9 o 

b. c. 

A ooo Bo§ 

Fig. 2. 
1, Types of spore- forming bacilli ; 2,, three types of spore germination * 
3, flagellse : (a) peritrichic, (6) lophotrichic, (c) monotrickic. 

Most bacteria are surrounded with a gelatinous, homogeneous 
covering termed capsule ; in some species — as the pneumococcus of 
Frankel and the pneumobacilli of Eriedlander — it may be easily 
demonstrated. It is not always present in cultivations, and is seen 
best in preparations made from the blood of an animal which has- 
succumbed to infection with the organism. The capsule is also 
seen in specimens obtained from pneumonic lung and "prune juice " 

Many organisms possess the power of independent movement 
and are described as motile. Upon such organisms extremely 



slender whip-like filaments may be observed by special methods of 
staining, known as flagella, and may be single and situated at one 
end, or the organism may be thickly studded with them ; they are 
often many times the length of the organism. Owing to their 
minute size it is difficult to say to which part of the bacterial 
cell they are primarily attached. With the exception of a few 
doubtful species motility is confined to the bacilli and spirilla. 
By examination of hanging drop preparations the motion can be 
easily observed, and the organisms seen darting about in all direc- 
tions. The absence of motility is not always synonymous with 
absence of flagella, many physical conditions causing " torpidity." 

Bacteria possessing flagella are classed as (a) Peritvichic, mul- 
tiple flagella surrounding the bacillus ; (b) Lophotrichic, tufts of 
polar flagella ; (c) Monotrichic, single polar flagella. (Fig. 2.) 

Fig. 3.— Involution Forms of Bacteria. 

Another form of movement which must not be confounded with 
the above is what is known as "Brownian movement," consisting of 
a dancing, oscillating swing of the organisms. It is a purely 
physical condition occurring with solutions of inorganic matter, 
exactly as with non-motile organisms, and is probably related to 
surface tension. In determining motility, observe the position 
of three organisms situated at the angles of an imaginary triangle 
and watch for a change of relative position, carefully excluding 
mechanical shock. 

Spore Formation. — Various organisms of the class bacilli pro- 
duce within their plasm highly refractile bodies which are capable 
of withstanding higher temperatures and stronger disinfectant 
solutions than the vegetative form ; these bodies are spores. 

The spores take up staining reagents with great difficulty, 
special treatment being necessary (see chapter on microscopical 
methods). Spore formation consists of a condensation of the cell 


plasm, the spore thus formed being surrounded with a tough mem- 
brane consisting of two layers. At the same time the remainder 
of the organism undergoes degenerative changes, eventually setting 
free the mature spore. Certain other clear spaces exist from time 
to time in the bacterial plasm, filled with lustrous drops of a fatty 
nature rendering observation alone useless in the determination of 
sporulation (page 47). 

The number of spores formed by a bacillus is rarely more than 
one, and is sometimes situated at one end, when the " drum stick " 
form is produced (Tetanus bacillus, fig. 2, 1). 

In some species (B. alvei), when the spore is centrally situated 
the cell plasm alters in form and becomes massed around the spore, 
forming a Clostridium or spindle-shaped mass. 

The form and size of the spores differ greatly in various species 
but is constant for the same species. B. subtilis has ellipsoidal 
spores 1-2 p. length by about 06 /* breadth. 

These spores formed in the interior of bacteria, or endospores as 
they are termed, are highly resistant to the action of heat, disin- 
fectants, or light, and a proper knowledge of them is of supreme 
importance in all bacteriological and hygienic work. 

Gruber and Brefield found that the spores of B. subtilis required 
three hours' boiling at 100° C. to kill them, whereas the rod-shaped 
forms were easily destroyed by heating for twenty minutes at the 
temperature of boiling water. When the spore is exposed to favour- 
able conditions it germinates, and in a short time (three to four 
hours for B. subtilis, Prazmouski) produces the parent form. The 
process is of three types : — 

(1) The spore gradually elongates, the outer membrane disap- 
pearing, eventually reaching the adult form which divides by 
binary fission, in the resulting cells spore formation again taking 

(2) The spore membrane is ruptured at the point of least resist- 
ance, and gradually grows out from the empty spore capsule, which 
dissolves in the surrounding fluid. The capsule, especially in motile 
forms, may be seen attached to the free-swimming rod. 

(3) The spore membrane ruptures at the equator of the spore, 
the developing rod gradually forcing its way out by one pole ; a 
portion of the spore may remain attached. 

Plasmolysis, or shrinking of the cell plasm with resulting spaces 
between the plasm and cell wall, may be produced by the action of 


certain reagents, among them being the ordinary physiological salt 
solution (0-75 gm. NaCl in 100 gms. of water). Various staining 
reagents bring about this phenomenon. The plasmolytic effect may 
be produced and removed without any apparent injury to the 
living cell. 


The Biology of Bacteria. 

Bacteria are greatly influenced by their surroundings, and on 
the other hand often profoundly modify the substratum in which 
they are growing, be it living tissue or nutrient solution, whilst the 
very products resulting from their activity are among the chief 
inhibitory influences restraining their indefinite development. 

Cohn estimated that a single bacillus 2 n long and 1 ^ broad, 
weighing 0-000000001571 mgrm., and which reproduced itself by 
binary fission once in half an hour, will in two days' time have a 
progeny of 281 billions, occupying a volume of half a litre, while in 
another three days the mass would be sufficient to fill the beds of 
all the oceans of the globe, the number of the progeny being repre- 
sented by 37 places of figures ! That such enormous development 
does not take place is due partly to the antagonism displayed by 
one species towards another, partly by the insufficiency of nutriment 
obtainable, but chiefly to the products of the organisms' own activity. 
The more important conditions related to the development of 
bacteria are — light, temperature, gaseous environment, moisture, 
food supply. The first four factors are related more particularly 
to the development of the bacteria, whilst the question of food 
supply is largely complicated by the various chemical changes 
induced by bacterial action. 

Effect of Light. — The antagonism of light to disease was a fact 
established by empirical observation long before bacteria were 
known to exist, and the old Italian proverb, " Where the sun does 
not enter, the doctor does," is illustrative of this popular knowledge 
gained by observation. Since the discovery of bacteria many 
experiments have proved the scientific basis of the empirical deduc- 
tion ; the majority of bacteria, and certainly all the known patho- 
genic forms, are particularly sensitive to the action of light. 

Direct sunlight is the most powerful agent ; exposure to the 


direct rays of the sun will kill tubercle bacilli in half an hour, 
anthrax spores in an hour. Diffused daylight has a similar, but less 
energetic effect, the time of exposure being about three times as 
long. The light from an electric arc has a similar effect to diffused 
daylight ; recently advantage has been taken of the fact in the 
treatment of lupus. 1 Tuberculous sputum and typhoid dejecta may 
thus be deprived of their respective organisms under natural con- 
ditions. Even when the organisms are not entirely destroyed 
by the process of insolation their pathogenic powers are greatly 
attenuated, the subsequent cultivations developing less luxuriantly 
than before the experiment, and the power of producing poisonous 
products greatly diminished. 

Among the bacteria producing pigment, light has marked effect 
in altering the power of chromogenesis. Thus if a cultivation of 
the B. rouge d'Kiel — an organism producing a fine red pigment — 
be exposed to sunlight for a time insufficient to entirely destroy the 
organisms, subsequent cultures may be obtained which have lost 
the power of colour-formation and remain colourless indefinitely. 
A few non-pathogenic bacteria, such as B. violaceous, which forms 
a purple pigment, apparently thrive best in the light. The red 
chromogens are generally more resistant to the action of light than 
other colour-forming species. 

Downes and others have attributed the destructive action of sun- 
light to a disengagement of nascent oxygen which attacks the 
bacterial plasma, such a process depending largely on the composi- 
tion of the medium coutaining the bacteria during exposure. 
The blue and violet portion of the spectrum, i.e., the most chemically 
active rays, were found most energetic in action, the red and yellow 
rays the least. The destruction was apparently independent of the 
temperature, and took place when the heat rays were excluded. 
The more translucent the medium the greater the action. 

Buchner found in a series of experiments conducted in the clear 
water of Lake Starnberg, that the bactericidal effect of light was 
apparent at the depth of two metres below the surface. Sunlight 
must therefore exercise a powerful influence in cleansing water- 
courses polluted with excremental matter ; the marked diminution 
in the number of bacteria present, say, a mile below a sewage 

British Medical Journal, January 1902. 


outfall, undoubtedly depends upon such action, besides sedimentation 
and other processes. 

Moisture. — Water is necessary for bacterial development as for 
other forms of life ; the optimum percentage of water is about 80 
per cent. Some bacteria will withstand desiccation for long periods, 
others rapidly succumb ; the spore-bearing organisms resist drying to 
a much greater degree than the non-sporulating varieties, the arthro- 
sporous forms holding an intermediate position (Hueppe). Anthrax 
spores will survive drying in dust for two years or more. A cultiva- 
tion of B. typhi abdominalis kept in my laboratory for nine months, 
and which had become so dry that the medium could be broken 
with ease, gave subcultures in twenty-four hours. 

B. diphtheria will resist drying for a week or two. Tubercle 
bacilli remain alive for long periods in rooms occupied by tuber- 
cular persons, especially in dark situations, as behind pictures, &c. 
On the other hand the cholera spirillum is destroyed by three to 
four hours' drying. 

Most Schizomycetes exhibit their maximum development upon 
fluid media, bacilli often growing out into long chains of filaments : 
" dry rot " and " mould " are always associated with dampness. 

Relation to Gaseous Environment. — Bacteria, in common with 
other living things, require oxygen for their existence, but not in 
all cases is it necessary that the gas should be present in the free 
state, as certain organisms can obtain the necessary oxygen 
from chemical compounds in which it is present in a loosely 
combined form. 

Such organisms are termed anaerobic, that is, they will live even 
though free air is excluded ; others however require oxygen in a 
free state, and are termed aerobic. Intermediate between these two 
extremes come those organisms which, although they develop best 
in the presence of air, are yet capable of existence when air is 
excluded ; such are termed facultative- anaerobic, and comprise the 
largest number of the known Schizomycetes. 

Anderobiosis is probably an ancestral trait going back to the first 
appearance of life upon this planet, when the atmosphere contained 
but little oxygen in a free state. It is possible experimentally 
to change the character of an organism that, though at first 
it is aerobic, subcultures will ultimately become anaerobic in 
habit, and vice-versa. Such experiments have been performed by 
Hueppe with the cholera spirillum. 


Hydrogen and nitrogen are indifferent gases for anaerobes, while 
sulphuretted hydrogen and carbon dioxide are poisons. On the other 
hand the majority of mouth bacteria are able to develop in an 
atmosphere of carbon dioxide — in fact certain species are favoured 
by its presence. 

The Beggiatoa group, as observed by Winogradsky, are able to 
exist in sulphuretted hydrogen, and change that gas into its com- 
ponent parts, storing up the sulphur in solid particles. 

The changes induced in the substratum by anaerobic bacteria differ from the 
changes taking place in the presence of free ox}-gen. The maintenance of life 
without free oxygen depends solely upon the availability of compounds from 
which oxygen may be split off. 

" The amount of chemical change therefore is relatively much less intense 
than in aerobic conditions ; thus if 1,000 grammes of sugar be completely oxi- 
dised to C0 2 and water in the presence of free oxygen, 3,939 calories or heat units 
are produced ; if however it is split into butyric acid, hydrogen and C0 2 , only 
414 calories are evolved. It follows therefore that anaerobic bacteria must 
superficially disintegrate a far larger quantity of material to obtain this neces- 
sary oxygen than aerobic organisms — a circumstance that has considerable 
significance in the large production of toxines by organisms growing in the 
living body. Again, during anaerobic fermentation the secondary products are 
not oxidised to other and simpler compounds, and they therefore accumulate, 
a good example being afforded by the so-called " bottom fermentation." 1 

It has also been shown by Fajans 3 that cholera cultures retain 
their virulence much longer under aniierobic than under aerobic 
conditions ; and Braatz :! has called attention to the fact that 
bacteria in suppuration foci are living without atmospheric oxygen. 

A great pressure of carbon dioxide is said to deprive B. anthracis 
of the power of sporulation. 

It is probable that facultative anaerobic organisms are largely 
concerned in dental caries after the granular layer has been passed, 
and the rapid progress and undermined character of the cavities 
generally formed is due to the phenomena connected with anaero- 
biotic growth ; gelatin, the end-product of the tooth cartilage or 
collogen, is a substance from which anaerobes are able to obtain 
their oxygen. A relatively large amount is therefore attacked, 
and in these anaerobic cavities much more of the matrix has 

Hueppe, " Princ. of Bact.," p. 54. 
Arch. f. Hyg., xx. 
1 Deutsche mcd. Wochcnsch., 1890, No. 4G. 


disappeared than is the case in those cavities to which oxygen 
has free access. 

Temperature. — Bacteria are the most widely distributed of all 
living things when we consider them from the point of view of 

Foster 1 and Fischer 2 have demonstrated that a number of 
bacteria, amongst them the species producing phosphorescence, 
thrive and multiply at 0° C. Per contra Miguel has described an 
organism that nourishes and forms spores at 70° C. (158° F.), a 
temperature at which ordinary albumen is coagulated ! These 
two extremes however only give the limits of actual growth, the 
limits of passive resistance being much wider. For instance, 
Pictet 3 found spores to resist exposure to the temperature of frozen 
oxygen ( — 213° C.) for a short time, and that they easily withstand 
a temperature of — 120° C. for twenty hours, rapidly developing 
when thawed. Eapid freezing and thawing was found to be more 
injurious than a long exposure to a low degree of cold. 

The ordinary bacterial plasm of most organisms enters into heat 
rigor at 42° to 45° C, and a prolonged temperature of 55° G. will 
destroy almost all bacterial bodies ; but the fact does not apply to 
the spores, those of B. subtilis requiring three hours' continuous 
boiling in w T ater or steam to destroy them. The point at which 
death occurs is termed "thermal death point," and varies con- 
siderably for various species. The property of resistance to tem- 
perature as high as boiling was one of the experiments by which 
bio-genesis was sought to be proved among others by van Helmont, 
who devised therefrom a process of producing artificial mice ! 

The spores present in the boiled fluid develop into adult forms 
as soon as the temperature has fallen sufficiently. From this, and 
the fact that the bacterial bodies themselves were easily destroyed 
by boiling, Tyndell devised what is known as intermittent steriliza- 
tion. The medium, which would be spoiled by a high temperature, 
is boiled for twenty minutes on three successive days. In the 
interval between the operations the spores germinate to adult 
forms which are killed at the next boiling. 

Saprophytic bacteria of soil and water grow best at about 20° C, 

1 Centralbl. f. Bakt, ii., 1887 ; xii., 1892. 

2 Ibid., iy., 1888. 

3 Arch, des Sci, Phys. et Nat., xxx., 1893, p. 293. 


growth ceasing at about 5° C. The pathogenic organisms have their 
optimum temperature at the body heat of about 37° C, although 
each of them generally exhibits a preference of some definite degree 
of heat which is termed " optimum temperature." 

The relative resistance of different bacteria is often made prac- 
tical use of to isolate the more hardy species. It is also used in the 
determination of the presence or absence of endospores. The 
cultivation or material to be tested is maintained at a temperature 
of 80° C. for half an hour. At the end of the time the tube is 
replaced in the incubator for twenty-four hours. If spores are 
present they resist the action of the heat and develop rapidly 
when incubated ; when no spores are preseut no development 

Heat is applied to " attenuate " pathogenic cultivations for 
inoculation purposes. Pasteur found that by incubating the anthrax 
bacillus at a temperature of 40° to 42° C. no spores are formed, 
and the pathogenic power of the bacilli is greatly reduced. Even 
spores themselves if maintained for considerable periods at 80° lose 
their pathogenic power. The digestive and bacterial enzymes are 
mostly destroyed by temperatures above 7CP, being more resistant 
than the vegetative forms but less so than the endospores. 

The foregoing facts have a very practical bearing upon steriliza- 
tion and will be again referred to under that heading : and in passing 
it may perhaps be as well to point out that freezing or cold-storage 
does not destroy the bacteria, but merely restrains their activity for 
the time being. 

Reaction of Medium.— Most bacteria grow best when the 
reaction of the substratum is neutral or faintly alkaline, the majority 
of the putrefactive bacteria and most of the pathogenic bacteria are 
favoured by an alkaline reaction ; some organisms, however, are 
able to grow on an acid medium, while a few are directly favoured 
by the presence of acid, as for instance the acetic acid bacilli, 
which ferment acetic acid to C0 o and water. B. butyricus is 
another of these acid-loving organisms. The mouth bacteria all 
prefer a somewhat alkaline medium, most of them refusing to 
develop in the presence of acid ; a few — particularly the mouth 
streptococcus— will grow in acid media. This ability of bacteria to 
develop in an acid medium must not be confounded with the 
production of an acid reaction by the vital activity of the organism. 
The reaction of the culture medium in which bacteria are 


cultivated, has considerable bearing on their development, different 
species showing a somewhat marked preference for certain per- 
centages of alkalinity — in fact, different races even of a given species 
of organism will show differences when grown on media con- 
taining slight differences in reaction, so much so that it is often 
possible to pick out a given race by these means. It follows, 
therefore, that in all practical bacteriological work definite and 
careful methods of standardisation should be adopted (see chap. 4). 

Food Supply. — The role of bacteria in nature is the breaking up 
of the complex chemical compounds of the bodies of plants and 
animals with the release of the chemical constituents so that they 
may be again recombined and utilised in building up fresh living 
things. A small section of the vast number of existing bacterial 
species have become so modified in their mode of life that they are 
able only to exist in the bodies of animals or plants, and developing 
in such situations initiate pathological changes with various symp- 
toms peculiar to special diseases. It follows, therefore, that the 
food material required by one species is not always adapted to the 
development of another ; in some cases — such for instance as the 
diplococcus of pneumonia, or the gonococcus — the organism can 
only be grown at first upon a medium smeared with fresh blood ; 
but even these refractory organisms in time adapt themselves to 
their new environment, and may be cultivated upon the ordinary 
laboratory media. The greatest number of bacteria, however, are 
remarkably adaptable, and may be cultivated upon what are termed 
" artificial media." 

An attempt is always made to reproduce, as far as possible, 
the natural food condition enjoyed by bacteria ; but to do so 
exactly is generally impossible owing to the complex nature of the 
natural food stuffs, and moreover to the frequent presence of 
organisms other than the special one it is sought to isolate. In 
a certain number of cases, however, the artificial cultivation may 
present better opportunities for the growth of a given organism 
than is possible in its natural habitat, many organisms excreting 
bodies harmful to other species and also to their own development. 
Two organisms therefore which, when growing together, exhibit 
mutual antagonism, may individually grow more easily when 
separated in pure culture in artificial media. 

Bacteria do not always antagonise one another, and many cases 
are known where the presence of one species of bacteria actually 


assists the development of another. Thus in the well-known 
fermentation that takes place when the juice of grapes is expressed 
furnishes an excellent example. The crude wine-must when it comes 
first from the press contains a large and varied flora, amongst which 
are yeasts. These yeast forms finding the surroundings especially 
fitted for their development ferment the sugar present to alcohol 
and COo until the alcohol reaches a certain percentage when they 
are unable to develop further. Another series of organisms now 
comes into play, contained like the yeasts in the original wine- 
must. These organisms attack the alcohol and change it to acetic 
acid, and as the alcohol becomes used up, cease their activity and 
give place to a third series, which having a special taste for acid 
solutions were unable to develop before their particular food was 
obtainable. As a result of their growth the acetic acid is fermented 
to COo and water, and the reaction of the medium becomes again 
neutral or faintly alkaline. The way is thus prepared for the 
putrefactive organisms which have gained access from the air, or 
from the original grape skins; these bacteria change the remaining 
proteid matters into CO.,, water, nitrogen and various evil-smelling 
gases that generally accompany putrefaction. Such a cycle is the 
common phenomenon in most spontaneous decompositions. The 
alcohol stage may be and often is omitted, direct change of carbo- 
hydrate into acid taking place. 

The whole process, one class of organisms clearing the way 
for the activity of another, is termed a " metabiotic cycle" or 
" mctabiosis" It often happens that two or more bacteria grow side 
by side and each assists the other, as for instance the B. tetanus, 
which is an anaerobic organism under ordinary circumstances, may 
be grown aerobically if the culture is also inoculated with B. pyo- 
cyaneus ; such a phenomenon is termed symbiosis, and is of great 
importance in many pathological conditions of mixed infection. 

Bacteria, as will be easily understood from the foregoing para- 
graphs, produce various chemical substances as the result of their 
growth ; some of these are due simply to the splitting up of the 
various molecules of food stuff into simpler parts ; the one is absorbed 
by the organism and built up into its protoplasm, the other remains 
in the solution. Other compounds are probably excreted by the 
organisms, and others again are only obtainable in any quantity 
from the bodies of the bacteria themselves. Some of these bacterial 
products are brightly coloured pigments, and it is easy to note in 


examining a cultivation of such a chromogen that the pigment pro- 
duced is not always confined to the actual area of growth of the 
organism but diffuses widely into the nutrient medium, demonstrating 
clearly the method in which the products of an organism taint the 
medium in which it grows, and in this way may check its own 
development (cf. B. pyocyaneus). 

The products of vital activity of bacteria are of many kinds, the 
whole of which are generally now included under the term " fer- 
mentation-products. ' ' 

These fermentations require considering in detail. 

Production of Acid and Alkali.— The majority of bacteria 
produce an alkaline reaction when grown in a medium free from 
carbohydrate ; some, e.g., the diphtheria bacillus, when grown in 
ordinary broth containing traces of carbohydrate, for the first few 
days give an acid reaction ; later the reaction changes to alkaline, 
often due to the presence of ammonia. 

Very many bacteria — and of these a considerable number are 
mouth bacteria — are capable of fermenting carbohydrate with the 
production of acid. The fermentability of various carbohydrates 
differs widely, glucose being the most easily fermentable. Lactose 
is also fermented to acid by mouth bacteria. The carbohydrates of 
the mono-saccharide group are those most easily acted upon, the 
general equation of fermentation being : — 

C 6 H 12 6 = 2 (C 3 H 6 3 ). 

Glucose. Lactic acid. 

The carbohydrates of the di-saccharide group, C 12 H 22 1:L , are 
first inverted to the mono-saccharide form and then fermented. 

Thus : + 

(i.) G^H^O^ + H 2 = C 6 H 12 0; + C 6 H 12 6 . 
Cane sugar. Dextrose. Lsevulose. 

(ii.) C 6 H 12 6 = 2(C 3 H 6 3 ). 

The carbohydrates of the poly-saccharide group are more complex 
than the other two groups, and require preliminary inversion before 
fermentation to acid occurs. Thus : — 

(C 6 H I0 5 )» + H. 2 = C 6 H lo 5 + C 12 H 22 11 . 

Starch. Dextrin. Maltose. 

C^H^O^ + H 2 = 2 (C 6 H 12 6 ). 
2(C 6 H 12 6 ) = 4(C 3 H 6 3 ). 


According to Brown 1 and Morris soluble starch has the formula 
(C 6 H 10 5 ) 30 . 

These equations do not express the whole of the reaction as 
some of the sugar is used by the bacteria themselves, and quantities 
of gas are often evolved during the process. 

In the formation of alcohol from sugar large amounts of C0 2 are 
evolved, thus : — 

6 H ia O a = 2C.,H,.OH - r 2 CO,. 

It has been shown by Maly- that proteid is not attacked to any 
appreciable extent as long as any carbo-hydrate remains in the 
solution, the organisms first attacking the carbo-hydrate, and only 
when this is used up is the proteid acted upon. 

Gas Formation. — Many bacteria produce gas, and a well known 
example is found in B. coli. com., which will produce large quantities 
of gas even in gelatin cultures. The chief gases formed by bacteria 
are CO.,, N, H, CH 4 , SH 2 . Unless the organism be anaerobic the 
gas formed generally escapes undetected into the air. When 
anaerobic a well-marked bubble may be seen around the colonies, 
as in stab gelatin cultures of Bacillus tetanus. In ordinary aerobic 
fluid cultures the formation of gas is best demonstrated by Durham's 
tube, a small test tube placed in the fluid, which becomes filled 
with the gas and floats in the liquid. 

Heat. — Some bacteria evolve a considerable amount of heat in 
their growth, and at times have been held responsible for spon- 
taneous combustion of vegetable matter (hay). Some of these 
organisms do not thrive below 4CP C, whilst they exhibit their 
greatest activity at 50 3 to 60^ C. Several species have been isolated 
and studied. Practically they are utilised in the formation of 
"ensilage," the heat evolved in the anaerobic silos being due to the 
fermentative activity of these " thermophilic bacteria." 

Nitrification. — The conversion of ammonia to nitrite and nitrate 
is accomplished by a number of bacteria, which are able to grow 
in purely inorganic material. These bacteria, which are present 
for the most part in the top layers of surface soil, are of great 
importance to the agriculturist. In artificial media a number of 
bacteria produce nitrates, the presence of which may be demon- 

1 J. Chem. Soc, Lond., 1888, 610. 

2 Hermann's Handbuch, Bd. v. (2) S. 239. 


strated by appropriate chemical means. Certain others of these 
nitrogen-loving organisms assist in fixing the nitrogen of the air. 

Phosphorescence. — A number of bacteria, many of them developing 
at 0° C, produce well-marked phosphorescence. Pfluger in 1875 
first demonstrated this relationship of bacteria to the silvery 
phosphorescence seen on unsound fish and meat. The amount of 
light produced has been shown to be sufficiently great to photograph 
small objects placed near the cultivation. The class, as a whole, 
show a well marked preference to certain food- stuffs ; in all cases a 
good supply of oxygen and about 3 per cent, of salt (NaCl) are 
required for the production of phosphorescence. 

Chromogenesis. — A considerable number of organisms, many of 
them belonging to the pathogenic varieties, produce various colour- 
ing matters, or pigments. The colouring matter may be confined 
to the bacteria themselves, or become diffused through the medium 
in which they grow. The pigments are often composed of several 
distinct chemical bodies which may be separated by chemical 
means. For instance B. pyocyaneus, the bacillus found in green 
pus, has been found to produce two if not three varieties of 
pigment. One may be easily obtained by extracting a cultivation 
with chloroform and crystallising out, when long delicate needles of 
a bluish-green tint are obtained, which changes to red on the 
addition of weak acid. The other pigment of B. pyocyaneus is a 
fluorescent green. 

B. prodigiosus, B. rouge de Kiel, and several bacilli found in the 
mouth, produce a fine red pigment. Two of the pyogenic cocci, 
Staphylococcus pyogenes aureus, and S. pyogenes citreus, produce 
well-marked pigmentation ; the colouring is not diffused into the 
culture medium. I have met with all the above bacteria in the 
mouth. Among other chromogens from time to time met with 
in the mouth Sarcina aurantiaca and Sarcina lutea are common. 
B. liquefasciens fluorescens, producing a fluorescent blue-green 
pigment, is also frequently met with. I have found this organism 
present in several cases of " green stain." 

The chromogenic function of bacteria is considerably modified 
by environment. Most chromogens only produce pigment when 
grown at a low temperature, 20° C, and it is often possible to 
artificially produce a variety of non-colour-producing organisms by 
simply growing them at 37° C. for several generations. For some 
little time after the organism has been subjected to the higher 


temperature it remains colourless, even when transferred to fresh 
tubes incubated at 22° C. In some species no colouration takes 
place at the low temperature ; the number is, however, limited to a 
few isolated examples. Thus the spirillum of Metchnikoff, when 
grown on potato at 37° C. forms a dark chocolate coloured layer, 
while at 22° C. no growth takes place. The bacillus of glanders is 
another organism producing colouration at the higher temperature 

The majority of chromogens require free oxygen for the elabora- 
tion of pigment, a few only producing their characteristic colour 
in the absence of air ; among these Spirillum rubrum may be 
mentioned. This organism is of interest, as Hueppe managed to 
so modify a race that the pigment was produced aerobic ally. 

Formation of Enzymes. — Many bodies belonging to the class of 
enzymes or ferments are produced by micro-organisms. 

Bacterial enzymes are of two classes : (a) those bodies which 
are freely soluble and are either excreted by the bacteria, or 
remaining in the bacterial plasm are easily dissolved out — inter 
cellular; (b) those ferments which are only obtainable by trituration 
of the bacterial cells, and not soluble under normal conditions — 

The enzymes produced by certain bacteria digest fibrin and 
gelatin when in an alkaline solution. These proteolytic enzymes 
may be separated from a culture of an organism by shaking up with 
chloroform water, filtering, precipitating with absolute alcohol, 
filtering and taking up the residue with sat. thymol water. The 
resulting filtrate will liquefy gelatin if the organism treated pro- 
duced a liquefying enzyme. In this way I have obtained extracts 
from various mouth bacteria which will digest decalcified dentine. 

Enzymes induce the various changes with which they are asso- 
ciated by a process of hydration or hydrolysis, that is by the addition 
of water to the body fermented, with the ultimate cleavage of the 
molecule into bodies of simpler chemical composition. 

These enzymes do not act well in an acid medium as do those 
of animal origin which react best in the presence of acid. 

Another class of enzyme produced by bacteria is the ferment 
allied to rennet bringing about coagulation of milk. 

A ferment, changing sugars of the disaccharide to the mono- 
saccharide form, is also formed by some bacteria, a few of which 
occur in the mouth. 


Putrefaction may be termed the fermentation of nitrogenous 
bodies by bacteria, and probably consists of a series of complicated 
changes occurring naturally by symbiosis. The first stage is a 
transformation of the albumin present to peptone, this being followed 
by the production of various gases, acids, bases and salts from the 

The bad smell of putrefying animal matter owes its origin to 
several members of the aromatic series, among which are indole and 
skatole, or j3. methyl indole. Indole combines with nitrous acid to 
form a red compound (nitroso-indole). Use is made of this in deter- 
mining the presence of indole in a cultivation. Those bacteria (e.g., 
cholera vibrio) which produce nitrite plus indole give the red 
colour on the addition of nitrite-free sulphuric acid. If no nitrite is 
formed, as B. coli, nitrite must be also added, either in a 0*3 per 
cent, solution of potassium or sodium nitrite, or by adding yellow 
(commercial) nitric acid, containing nitrites. This indole reaction 
without the addition of nitrite is often known as the cholera red 
reaction, as it was first described in connection with that organism. 

Phenol, ortho- and para-cresol, leucine and tyrosine are also- 
formed by the action of putrefactive bacteria. 

Sulphuretted hydrogen is commonly formed among other gases* 
and is recognised by adding an iron compound (iron-lactate) to the 
culture medium. 

Ptomaines. — Various poisons are formed by the decomposition of 
putrefying albuminoids, some of which produce serious symptoms 
when ingested by man. The substances often result from the 
growth of organisms in various articles of food, among which may 
be mentioned tyrotoxican from the decomposition of cheese, and 
hydrocollidine from the flesh of cattle. 1 

It occasionally happens that putrefaction with the formation of 
similar poisonous bodies may go on in the intestinal canal, the 
products in such case receiving the term leucomaines , and are 
probably the chief cause of the headaches so often associated with 

Many putrefactive bacteria are obtainable from the mouth ; 
especially is this the case in individuals possessing unclean 
mouths and many decomposing roots. 

The ptomaines are definite chemical bodies which have been 

1 Vaughan and Noug, "Ptomaines and Leucomaines." 


isolated by Briger and others and their percentage composition 
determined. The toxines (see below) have not yet been isolated in 
a chemically pure form. 

Toxines. — A number of pathogenic bacteria produce poisons 
during the period of growth, and the symptoms of certain diseases 
are due to the absorption of these toxines. These bodies have not 
yet been isolated in a true chemical form, but by filtering a broth 
cultivation in which a toxine-forming organism has been grown the 
bacteria are filtered off, and the germ-free filtrate contains the 
toxines. This solution of toxine injected into susceptible animals 
produces death. The filter used is of unglazed porcelain (see 
fig. 11). By evaporating the filtrate to one-third at 30° C. in vacuo, 
precipitating by alcohol, again taking up in water, and repeating the 
process several times, a white powder may be obtained from the 
filtered culture of diphtheria. This white powder injected into 
guinea pigs produces the same symptoms as injection of living 
diphtheria bacilli. 

Sidney Martin 1 isolated from the cultivations of diphtheria, as 
well as from the spleen, &c, of patients dead of the disease, two 
bodies, one of the nature of an albumose, the other an acid. These 
substances when injected into animals produced the same symptoms 
as diphtheria toxine. There was, however, a certain difference due 
to the fact that the albumose isolated behaved as a digestive enzyme, 
forming the true toxine from the body tissues. Smaller but repeated 
doses produced more marked effect than single large doses ; the 
natural conditions of diphtheria poisoning, consisting of gradual 
absorption rather than sudden intoxication, were thus copied. 

Toxic bodies may be prepared from cultivations of tetanus, 
typhoid, staphylococcus aureus, cholera, &c, by the method adopted 
for diphtheria toxine. 

The fluids obtained by filtration, &c, or the amorphous alcohol 
precipitate both show similar reactions, that is : (a) on injection 
into animals ; (b) a temperature of 58° C. for two hours destroys 
the pathogenic properties. These toxines are classed under the 
group of intercellular toxines, destruction of the bacterial cells not 
being necessary to obtain the poisons, which are freely soluble in 
the liquid media in which the bacteria are grown. On the other 
hand many organisms, if not all, possess poisonous properties 

1 Local Government Board Reports, 1891. 


within their own plasm or micro-protein. The material used in 
"vaccination" for typhoid fever consists of such intra-cellular 
poison of the typhoid bacilli. 

The inter-cellular and more soluble toxines appear nearly related 
to the digestive enzymes of animal glands, such as trypsine and 
pepsine in their method of action, and it is extremely probable 
that the nerve degeneration of diphtheria and the solution of fibrin 
by digestive ferment proceed along exactly comparable lines. Under 
such an hypothesis it is easy to understand why a small continued 
dosage of a given bacterial poison will produce such profound effect, 
and how it comes about that such minute quantities are relatively 
so potent. Probably the change is the same process of hydration 
that we have seen occurs in the carbohydrate transformation, and 
that when the molecule has become enlarged by the addition of 
water to a given extent it breaks up along new planes of cleavage. 

It is of course possible that the true toxic bodies are definite 
chemical compounds which are precipitated along with the albumoses 
in the alcohol method adopted. So far, however, all attempts to 
obtain definite crystalline bodies have failed, and all we are able 
to state is that the toxine, whatever it may be, is found in the 
precipitate thrown down by alcohol from cultivations of bacteria- 
forming toxines, and that the precipitate thus found certainly 
contains albumoses. 

For further information on toxines see chapter on immunity. 


Sterilization and Disinfection. 

Bacteria are the most widely distributed of living things ; they 
teem in the dust of cities, in hospital wards, they are to be found in 
countless numbers in the soil, in the air we breathe, in common 
articles of food, in water, and particularly in the dusty air of streets 
and living rooms. 

The air of high mountains and mid-ocean are generally practically 
free from organisms, whereas city air may contain as many as 
100,000 or more per cubic foot. They have recently been found in 
glacier ice. 

The organisms present in air are by no means all pathogenic, 
but at the same time many pathogenic bacteria are frequently 
present ; amongst them the pyogenic cocci are common. The source 
of the organisms in the air is for the most part dust, and where dust 
contains the dried expectoration of tuberculous persons the tubercle 
bacillus is invariably present. During damp and wet weather the 
number of organisms present diminishes considerably, the falling 
rain freeing the air from suspended matter and bacteria, which are 
carried away with the surface water in properly drained places, or 
remain in the mud of pools to be wafted into the air as dust when 
the water evaporates. 

Bacteria of the air are, for the most part, simple saprophytes, 
and although not disease-producers in the ordinary way are capable 
of setting up profound changes in organic fluid exposed to their 
advent, producing " disease " in such articles as milk, meat, &c. 
Many of the spores of the higher fungi are air-borne as well as 
yeasts and torula. A gelatin plate exposed to the air for a few 
moments will generally develop a number of colonies when incubated. 
I have already referred to the minute size of these micro-organisms, 
and it is not difficult to understand that almost anything with which 
we commonly have to deal in bacteriological work is contaminated 


with numbers of unseen organisms ready to develop the moment we 
make the conditions favourable for them. And what is true of 
bacteriological apparatus is still more true of dental and other 
instruments, for with these latter not only are air-borne organisms 
present, but also those from infected wounds, oral secretions and 
decaying dentine, septic pulps, &c, with which they have been in 

Owing to their minute size bacteria are carried about by the 
slightest currents and motion of the air, but in still air they gradually 
sink to the lower strata. Tyndall proved that when the dust in 
a specially constructed room had been allowed to settle till the 
polariscope showed no trace of suspended matter, sterile open vessels 
of nutrient solution could be freely exposed without decomposition 
taking place as long as the dust remained quiescent. When, how- 
ever, the dust was again made to rise the fluids quickly became 

It is evident that any materials with which we wish to conduct 
bacteriological experiments must be first of all freed from the 
organisms naturally present, otherwise we shall be unable to 
determine if the particular fermentation or growth we are examin- 
ing is the product of a single species or of a mixture of species, or 
in mycological parlance know if we are dealing with a pure culture. 

Pure cultures, consisting of members of one given species only, 
are the means by which determinative bacteriology has been 
rendered possible. Although much of the earlier work was con- 
ducted with what are now known to have been mixtures, it must 
not be supposed that the combined action or symbiosis of bacteria 
is to be disregarded, many of the most interesting of natural 
fermentations belonging to symbiotic phenomena. 

But to properly study the combined activity of two or more 
bacteria we must first have pure cultures of each from which to 
make our mixture. A considerable portion of bacteriological 
technique is directed towards obtaining pure cultures, and the 
process of excluding adventitious organisms is termed sterilization. 
Heat in some form is commonly used, of such a temperature that 
the articles sterilized are not injured while the organisms present 
are killed. The difference in resisting power has been already noted, 
and the reader is advised to take particular note of the "resisting 
power " of various organisms, as it has large practical bearing upon 
the question of sterilization. 



Heat is applied in two forms : (1) Dry heat ; (2) Moist heat. 

(1) Sterilization by Hot Air. — The various pieces of apparatus 
used in bacteriological work, such as flasks, test tubes, Petri dishes, 
and the like, are sterilized by heating to 150° C. for three-quarters 

F IG< 4, — Hot-Air Sterilizer with Apparatus ready for Sterilization. 

Fig. 5. — Copper Box and Back for Sterilizing Petri Dishes and Capsules. 

of an hour in a hot-air sterilizer; the flasks, &c, are first plugged 
with cotton wool plugs, which so long as they remain dry prevent 
the passage of bacteria. The hot-air sterilizer (fig. 4) consists of a 



copper or sheet iron box with hollow walls and a fire-brick bottom, 
placed upon a stand to admit of a large gas burner underneath. 
There is a hinged door opening the whole width of the sterilizer. 
In the roof are two tubes communicating with the inner chamber, 
through which a thermometer is placed to register the temperature 
of the interior. 

Petri dishes, capsules (figs. 6 and 7) small Petri dishes, 5 cm. 
wide), pipettes, &c, are placed in special copper boxes with a 
central movable rack from which the plates may be lifted out when 
required (fig. 5). 

Fig. 6. — Petri Dish. 

Fig. 7. — Glass Capsule. 

Test tubes, flasks, &c, are first plugged with cotton-wool. A 
piece of wool is folded up and twisted into a firm plug and forced 
into the mouth of the tube, about a third left projecting. The test- 
tubes are placed in wire crates which fit into the sterilizer. 

A convenient addition is a " contact alarm," so arranged that 
a bell rings when the temperature reaches the point required. 

The temperature is allowed to rise slowly to 170° C, when the 
gas is turned out and the apparatus allowed to cool down. The 
door must not be opened till the temperature has fallen to 60° C. 

The temperature here suggested is that which is found to 
destroy spores, the vegetative forms succumbing at a much lower 
temperature (68° C). For the various liquid and solid media used 
170° C. is too high, and would evaporate and char the tube contents. 
Streaming steam in the steam sterilizer is therefore used. 

(2) Sterilization by Streaming Steam. — Although the spores of 
most bacteria resist the application of 100° 0. for a considerable 



time, yet the vegetative forms are destroyed at relatively low 
temperatures. Having this in mind Tyndall suggested the discon- 
tinuous method of sterilization by streaming steam. The operation 
is generally carried out in a Koch's or other steam sterilizer. 
Tyndall found that although the spores were not killed by twenty 
minutes' steaming the vegetative forms were, and therefore if twenty- 
four hours were allowed to elapse after the first heating, any spores 
present would germinate in the nutrient media, and be easily 
destroyed by a subsequent heating. This is the method generally 

Fig. 8. — Koch's Steam Sterilizer. 

adopted. The media is placed in the test tubes which have already 
been sterilized in the hot-air sterilizer and placed in the steamer 
for twenty minutes on three successive days, after which the media 
is ready for use. 

The steam sterilizer (fig. 8) is simply a modified potato steamer 
or double saucepan, with an asbestos jacket to minimise radiation. 
The tubes should not be placed in the apparatus until steam is 
given off, otherwise considerable condensation takes place , and for 
the same reason should be removed as soon as sterilized. 



(3) Steam under Pressure is also made use of in various ways, 
and is the method generally adopted for the disinfection of articles 
of clothing, bedding, &c. The autoclave (fig. 9) is the apparatus 
used in the laboratory, and consists of a strong copper boiler with 
removable lid, which can be adjusted by means of a series of thumb 
screws set at intervals. There is a pressure gauge, thermometer 
well, and safety valve in the lid. 

Water requires a pressure of 15 lbs. to the square inch to boil at 
100° C., and 15 lbs. extra, that is, two atmospheres, to boil at 
115° C. The safety valve is set to blow off at 115°, and the medium 
sterilized for fifteen minutes at this temperature. 

Gelatin must not be sterilised in the autoclave as a considerable 
amount of hydration to gelatin peptone often occurs, impairing the 

Eig. 9. — Autoclave. 

Value of the medium, which may subsequently refuse to set ; even 
when the temperature does not rise above 105° C. peptonisation 
will occur, and long boiling may also produce the same effect. 
The peptonisation of gelatin by autoclave sterilization was particu- 
larly impressed upon my mind when attempting to sterilize gelatin 
in bulk for some physiological experiments ; two litres of 20 per 
cent, gelatin were placed in the autoclave at 115° for twenty 
minutes, but on cooling to the ordinary room temperature afterwards 
the whole quantity refused to solidify — the gelatin was entirely pep- 
tonised. Milk may be conveniently sterilized in this way ; agar 
generally darkens considerably, and had better be sterilized by the 


discontinuous method. Broth may be autoclaved, as may potato, 
but all these media are best sterilized by streaming steam. 

(4) Sterilization at Low Temperatures.— The vegetative forms of 
most bacteria are easily destroyed at low temperatures (55° — 60° C). 
Advantage is taken of this fact in sterilizing blood serum and other 
fluids which coagulate at 75° — 100° C. The serum collected under 
aseptic precautions is kept at a temperature of 57° — 58° C. for an 
hour on five or six successive days. The temperature adopted does 
not coagulate the serum, which may be used in the fluid condition 
if desired, and more important still may be kept in sterile flasks of 
convenient size for indefinite periods, care being taken to avoid 
certain infection of the plugs with air-borne spores. Koux's paper 
caps maybe advantageously used, or better still " sterilized milk " 
bottles or rubber caps may be placed on the tubes. 

Fig. 10. — Koch's Blood Serum Inspissator. 

Sterilization of Instruments. — The various instruments used for 
post-mortem examinations, injection syringes, &c, are sterilized 
by boiling in water in a suitable copper vessel fitted with a per- 
forated tray. A small quantity of carbonate of soda is added to the 
water to prevent rusting. A quarter of an hour is generally con- 
considered sufficient exposure to boiling water for all practical 

Dressings, bandages, and the like, may be sterilized in the auto- 
clave, or by the hot air method ; the former is preferable, dry air 
being afterwards passed through the apparatus. 



The platinum wires used in the inoculation of media during the 
process ,of making cultivations, forceps and various other small 
articles that are not injured by heat are sterilized by heating in the 
bunsen flame. The platinum or platino-iridium inoculating needles 
must be heated in the flame till red hot before and after use, to 
prevent the contamination of the culture or the dissemination of the 
organisms in the culture tube. The platinum needle is first heated 
to redness and then the glass or aluminium handle passed through 
the flame also. It is essential that no wire should be laid down 
under any consideration whatever without previous sterilization. 

It is not perhaps out of place to note here that the majority 
of dental instruments may be sterilized by boiling with water con- 
taining 1 per cent, of sodium carbonate, in the manner adopted for 
other surgical instruments. 

With some of the finer instruments it is better to substitute pure 
almond oil for the water in a small sterilizer j the edge and temper 
are not affected in the least. 

Fig. 11.— Unglazed Porcelain Filters (Pasteur-Chamberland). 

Sterilization by Filtration. — This method is largely adopted in 
the preparation of the soluble products of bacterial activity, such as 
toxines and enzymes. 

The material, broth cultures, for instance, is placed in a 
specially constructed hollow cylinder of unglazed porcelain (fig. 11). 
The cylinder is fitted into the mouth of a sterile filter flask by means 
of an india-rubber washer previously sterilized by boiling, and nega- 
tive pressure developed by means of a filter pump. The canals of the 
porcelain are so minute and tortuous that the fluid alone can pass 
through, the bacteria being arrested. To sterilize the " filter candle " 
after use it may be heated to redness in a muffle, using great 


caution, or hot alkaline permanganate solution may be filtered 
through, by which means the bacteria remaining in the canals of 
the filter are dissolved. Hydrochloric acid may also be used, but 
considerable care is required to wash away the acid afterwards. 

Many fluids may be prepared for cultural purposes by filtration 
in this way if the process of sterilization at 100° C. damages them. 

Various forms of water filters are constructed of unglazed 
porcelain and form the only efficient bacteriological filters. These 
filters do not however work indefinitely, as in about a week the 
bacteria which are arrested by the windings of the canals have 
grown to such an extent that the filtrate becomes contaminated. 


The terms antiseptic and disinfectant are somewhat misleading 
in that a substance which will certainly destroy bacteria or their 
spores in a fairly strong solution (disinfectant) will only inhibit their 
growth when used in higher dilutions (antiseptic). It follows then 
that antiseptics only hinder the growth, while disinfectants destroy 
the life, of bacteria. 

A large number of chemical substances have been used from time 
to time, many of these substances eventually proving to be of little 
value, not perhaps so much on account of the inefficiency of the 
chemical to destroy bacteria as on account of the wasting of the disin- 
fectant by reason of other substances present. It may often happen 
that owing to the presence of some body with which the disinfectant 
easily forms compounds — such for example as mercuric chloride and 
albumin — a large quantity of the supposed disinfectant is rendered 
inert by precipitation. Permanganate of potash readily oxidises all 
organic matter, whether bacteria or proteid compounds ; it is there- 
fore necessary in choosing an antiseptic to obtain the most efficient 
one for the special purpose for which it is to be used, having regard 
to the particular local conditions. It must also be remembered that 
many antiseptics and disinfectants are at least as injurious to the 
cells of the body as to the bacteria they are employed to destroy, and 
a solution used in such strength actually favours the entrance of the 
organisms by lowering the tissue vitality. This effect of antiseptic 
solutions is often overlooked, and it follows that a great deal more 
may be done by preventing the access of organisms than by attempt- 
ing to destroy them when they have once gained a footing. 


The following list of antiseptics and disinfectants gives some of 
the more- common ones in use with their relative strength as deter- 
mined practically by laboratory experiments. These tests, however, 
are more favourable to the antiseptic used than the organism tested, 
which is growing artificially and not in its usual habitat. The use 
of spores and their death as determined by absence of germination 
is more reliable; the spores to be tested are dried upon sterile 
silk threads and immersed for various periods of time in the anti- 
septic to be tested, then washed with boiled distilled water to 
remove traces of antiseptic and transferred to a culture tube. 

Another method is to add various quantities of the antiseptic 
under investigation to broth cultivations of the organisms experi- 
mented with ; in this case care must be taken to avoid fallacies due 
to the neutralisation of the antiseptic by the medium used. The 
cultivations may be either fully developed ones, or one or more 
loop-f uls (ose) of culture may be inoculated into the nutrient medium 
containing the antiseptic to be tested. In making the subsequent 
sub-cultivations to test the destruction or inhibition of the organisms 
care must be taken to use a sufficiently large quantity of nutrient 
medium, otherwise the amount of antiseptic in the ose may invalidate 
the result. Control tubes should invariably be made. 

Sternberg recommends mixing the standard culture and the 
diluted antiseptic in equal proportions ; thus 10 cc. of sterile broth 
containing 1 in 200 carbolic is added to 10 cc. of a twenty-four hours' 
broth culture of the given organism ( = 1 in 400) ; plate cultivations 
are then made at given intervals. 

Many substances, such as concentrated solutions of sugar or 
common salt, prevent the development of bacteria but do not kill 

Disinfection of Hands, &c. — The bacterial flora of the skin 
is of a varied nature, and owing to the cracks and fissures of 
the epidermis, particularly the hands, it is difficult to remove the 
bacteria ; moreover the bacteria actually live upon the dead 
epithelial cells, rarely however penetrating the true skin. The 
best method to adopt is first thorough scrubbing with a nail brush 
(boiled and kept in 2 per cent, lysol), soap and hot water, to 
remove as much of the dry epidermal scales as possible. The hand 
should be then soaked for two minutes in some antiseptic solution 
such as the one recommended by Lockwood 1 , 1 of biniodide of mer- 

1 Brit Med. Jour., Jan. 11, 1896. 


cury in 500 of methylated spirit, which is subsequently washed off 
in 1 in 3,000 biniodide solution. This treatment does not cause 
the same roughness that mercuric chloride or carbolic so often 
produces. It need hardly be added that unless a perfectly sterile 
towel is used the disinfection is of no avail, and in operative surgery 
they are discarded entirely, and should not be used by the dental 
surgeon to dry his hands before the operation of extraction. 

The following short list of antiseptics and disinfectants gives the 
most useful ; for other and extensive lists the reader is referred to 
Sternberg's "Bacteriology," Macfarland's "Pathogenic Bacteria," 
and Hueppe's exhaustive article in " Principles of Bacteriology." 

Formalin (40 per cent. sol. of formic aldehyde gas in water) ... 1 in 40,000 

Biniodide of Mercury ... ... ... ... 1 „ 35,000 

Bichloride of Mercury ... ... ... ... 1 ,, 14,000 

Lysol ... ... ... ... ... ... 1 „ 1,000 

Carbolic Acid ... ... ... ... ... 1„ 133 

These figures give the relative strengths of solutions which will 
restrain the growths of bacteria but will not always destroy them. 

Salicylic acid and quinine are also powerful antiseptics, whilst 
iodoform, so commonly used, must be first changed into iodine — a 
somewhat rare thing — before it is effective. Its chief action is the 
neutralisation of the products of the organisms. The various mineral 
acids are strong disinfectants, and 5 per cent. HC1 added to 
mercuric chloride greatly increases its efficiency. Lysol, consisting 
of coal tar oil, phenol and soap, is advantageous in that it is strongly 
alkaline and dissolves grease. It is used largely in general labora- 
tory routine, especially for soaking used slides, the coverslips 
becoming detached by solution of the balsam as soap. A jar 
containing a 2 per cent, solution should be kept on the laboratory 


Methods of Observing Bacteria — Microscopical. 

The microscopic examination of bacteria is carried out in two 
ways : (1) observation of living organisms ; (2) stained preparations. 

(1) Observations on Living Bacteria. — A small tin ring is 
cemented on to a glass slide with Canada balsam, forming what is 
known as a hanging-drop slide (fig. 13). A clean coverglass (see 

Fig. 12. — Coverglass Jar for keeping Coverslips in Alcohol. 

appendix) is removed from the jar (fig. 12) and the alcohol burnt 
off ; a drop of water placed in the centre by means of the platinum 
loop, and the drop inoculated with a minute amount of the culture 
to be examined ; the ring on the hanging drop slide is smeared 
with a little vaseline by means of a small paint brush, and the 
coverslip placed upon it drop downwards ; the vaseline prevents the 
coverslip falling off and keeps the preparation from evaporating. 
The preparation is now ready for examination, and is placed under 
the microscope and examined first with the J. Motility, Brownian 
movement and spores should be looked for; the spores, if present, 
appear as highly refractile bright dots. 


To watch the development of an organism under the micro- 
scope some sort of warm stage is required, a constant temperature 
being maintained by means of circulating water and a thermo- 
regulator. The hanging drop is used, with a nutrient solution 
substituted for the water. 

The hanging-drop method is used for the determination of the 
agglutinating power of serum, as in Durham's (Wedl's) typhoid 
reaction and spore germination, chemiotaxis, and many other 
experiments with living organisms. Every organism should be 
submitted to this process besides the methods of staining given 

Fig. 13. — Hanging Drop Slide. 

(2) Coverslip Preparations.— In coverslip preparations the bac- 
teria are fixed on the coverslip, and stained by one or other of the 
various stains given below. 

A coverslip is taken, and when the alcohol has been removed, 
a drop of sterile water is placed in the centre. With a sterilised 
platinum wire, sterilised by heating to redness in the flame, a small 
quantity of the culture to be examined is removed and added to 
the drop of water. Only a small amount is used, otherwise the 
preparation is too thick and the individual organisms massed 
together in clumps. A faint cloud throughout the drop is all that 
is required. The drop now containing the bacteria is carefully 
spread over the surface of the coverglass and allowed to dry. 

When dry, but not before, the coverslip is "flamed." To do 
this take the coverslip between the finger and thumb and pass 
downwards through a bunsen flame, repeating the process three 
times. There is nothing mystic in the " three times through the 
flame," but it has been found by the experience of a large number 
of workers that this fixes the bacteria properly without damaging 
them for staining afterwards. After a little practice the student 
will be able to hold the coverslip in forceps, but it is far better to 
use the fingers at first until the method is mastered. After cooling 
the coverslip is flooded with stain. A good method is to use an 


indiarubber coin pad such as is used for " change " in many shops ; 
the slips can be easily manipulated in this way. In some processes 
it is better to immerse the coverslip in a watchglass full of stain. 

When stained the preparation is well washed in water, dried 
between folds of blotting paper, and may be finally dried a safe 
distance above the flame (one foot). The preparation is then laid 
film upwards upon a piece of blotting paper, a drop of Canada 
balsam dissolved in xylol placed in the centre, and a clean slide 
pressed upon it; the blotting paper absorbs any excess of balsam. 
The preparation is now ready for microscopic examination. A drop 
of cedar oil is placed on the coverslip and the oil immersion lens 
lowered until it touches the oil, and all but touches the glass ; 
great care must be exercised to prevent the lens actually coming in 
contact with the glass, otherwise it may be irreparably damaged. 
To find the focus, rack upwards with the coarse adjustment until 
the film comes into view and then use the fine adjustment. 

Films from Liquid Cultures. — The films made from broth or 
from the mouth direct contain a considerable amount of material 
which stains as well as the bacteria, forming an undesirable back- 
ground. To prevent this the film must be " cleared " with some 
solution which does not interfere with the later processes of 

The films may be cleared in 1 percent, acetic acid, or in absolute 

Another method of fixing film preparations suggested by Gou- 
lard is as follows : the coverslips, dried in air but not flamed, are 
immersed in a solution of absolute alcohol 25 ccm., pure ether 
25 ccm., alcoholic solution of mercuric chloride 20 per cent. 0-5 ccm. 

The films are left in for five minutes or longer, washed well in 
water and stained. 

Tissue Preparations. — These preparations may be fresh, the 
tissue being cut with the freezing microtome, or fixed and hardened, 
and cut with the rocking or other microtome. 

Fixation. — Small pieces of tissue may be hardened and fixed at 
the same time in absolute alcohol. Corrosive sublimate, a saturated 
solution in 0-75 per cent, sodium chloride solution is very useful ; 
pieces £ in. in size or less are left in solution for twelve hours, larger 
pieces for a longer time. After fixing they are placed in a gauze 
bag in running water for twenty-four hours, and then passed through 
three percentages of spirit, 30 per cent., 60 per cent., 90 per cent. 


Some iodine is added to the 60 per cent, to remove the last traces 
of the mercury salt. 

Embedding. — The various stages in the process are as follows : — 

(1) Preliminary fixation and hardening as above. 

(2) Absolute alcohol to complete dehydration. 

(3) Absolute alcohol and xylol, equal parts, for twenty-four hours. 

(4) Xylol twenty-four hours. 

(5) Xylol and paraffin twenty-four hours in paraffin bath. 

(6) Pure paraffin three or four hours. 

(7) Melted paraffin poured into mould composed of two L- shaped 
pieces of brass, and just before it sets the tissue placed in it. 

(8) Trim up when hard and cut on microtome. 

Preparation of Sections of Teeth to show Bacteria in situ. — 
Owing to the leathery consistence of dentine when decalcified, 
the specimens cannot be cut in paraffin with any degree of success, 
and celloidin is generally employed. 

Care must be taken in decalcifying, otherwise the bacteria do not 
stain well ; the best agent is trichloracetic acid, suggested to me by 
Dr. Spriggs, which gives admirable results. 

The tissue is fixed in Goulard's solution or other fixative, washed 
and transferred to the acid (5 per cent, solution) till soft. 

When thoroughly softened the preparation is well washed, 
dehydrated and embedded in celloidin in the usual manner. The 
sections are stained after cutting. Carious dentine, &c, may be 
embedded in gum while fresh and cut when frozen. 

To prepare and stain the sections obtained by the paraffin 
method the embedding process is reversed. 

Float the section in warm water on to a clean slide and dry. 
Eemove the xylol with absolute alcohol, the alcohol with water. 

The specimen is now stained, washed rapidly in water, then 
alcohol, and finally xylol, and mounted in Canada balsam dissolved 
in xylol. 

Blood Films. — Method I. — Place a drop of the blood to be 
examined upon a clean slide near one end. Take a second slide 
and place the edge in the drop so that the whole of it becomes 
wetted, then push the slide along the surface of the first, keeping 
the second slide inclined. 

Method II. — Moisten the edge of a cigarette paper with the blood 
to be examined and quickly smear the slide or coverslip. Two or 
more coverslips may be held in a clip or on a piece of blotting paper 
by means of a slide. 


These films require special treatment if the corpuscles are to be 
preserved. The films are fixed by one of the following methods : — 

(1) In a hot air oven at 120° C. for an hour. 

(2) In equal parts of alcohol and ether for half an hour. 

(3) In saturated mercuric chloride solution for three to ten 

After fixation the films are washed, stained, dried and mounted. 
For special stains for blood films the reader is referred to the 
large text-books. 


General Principles. — The stains generally used in the laboratory 
for the staining of bacteria belong to the aniline series of basic 
nature, the bacterial plasm staining much in the same way as the 
nuclear chromatin of animal cells. 

The aniline dyes are divisible into two series according to whether 
the acid or basic part of the dye is concerned in the process of 
staining. The basic stains are the ones that have the greatest 
affinity for the nuclear chromatin and bacteria, the acid for the 
protoplasm of the cell. 

The following aniline dyes are among the ones commonly used 
in mycological work : Aniline gentian violet ; dahlia (methyl-violet) ; 
methylene blue (phenylene blue) ; methyl green ; thionin blue ; 
Bismarck brown (vesuvin) ; fuchsin (basic rubin). 

General Remarks on Stains. — The red and violet stains are the 
most intense in their action, and it is particularly easy to overstain 
with them ; they are also liable to form " background." Specimens 
stained with gentian violet may often be cleared in absolute alcohol 
without decolourising the organisms. 

The two blue stains are not so intense in their action but give 
more detail of structure; methylene blue particularly is useful in 
this respect, many appearances of the protoplasmic contents of the 
organisms being only demonstrable by its use. They are largely 
used for general routine work and for counterstaining for contrast. 

Stock solutions of the above stains are conveniently kept in 
alcohol ; a quantity of the stain is placed in a glass-stoppered bottle 
and alcohol poured in, as it is used from time to time the bottle is 
filled up with fresh alcohol. 

Watery solutions of the stains are also used and may be kept 
made up, 1 per cent, being the usual percentage. 


All stains require filtering, as decomposition occurs with the 
formation of granules which become deposited on the specimen ; 
the violet and red stains are particularly liable to do this. 

Many, if not most, stains work better and with more rapidity if 
some mordant be also added to the solution ; among these mordants, 
carbolic acid, aniline oil, and caustic potash are severally employed. 
Carbolic is the mordant commonly used ; a 5 per cent, watery solution 
is kept made up and only mixed with the alcoholic solution of dye 
immediately before use. 

In some staining processes decolourising agents are employed 
to differentiate between certain bacteria, some remaining unaffected, 
others are decolourised (Gram's method). 

Fig. 11. — Stand to hold Bottles of Stains, &c, for Laboratory Bench. 

Formulae of Stains. 

(1) Lofflcfs Methylene Blue. 

Saturated alcoholic solution methylene blue . . 30 cc. 

Potassium hydrate (1 in 10,000 in distilled water) . . 100 cc. 
Very little liable to overstain, even when left in contact for a 
long time. Eosin may be used as a counter-stain. 

(2) Carbolic Methylene Blue. 

Methylene blue . . .. .. .. .. .. 1*5 gm. 

Absolute alcohol . . . . . . . . 10 cc. 

Carbolic acid solution (1 in 20) . . . . . . 100 cc. 

Carbolic Thionin Blue. 

Thionin blue . . . . . . . . . . . . 2 gm. 

Carbolic acid solution (1 in 20) . . . . . . 100 cc. 

(S) Aniline Gentian Violet. 

(i.) Saturated alcoholic solution of gentian violet. 

(ii.) Saturated watery aniline oil (aniline water). 

The aniline w T ater requires filtering, and should be freshly prepared 
each time by shaking up 5 cc. aniline oil with 200 cc. distilled 
water. Immediately before use 1 part of the stain is added to 10 
parts of the aniline water. 


(4) Carbol-Fuchsin. 

Fuchsin . . . . . . . . . . . . . . 1 gm. 

Absolute alcohol . . . . . . . . 10 cc. 

Carbolic solution (1 in 20) 100 cc. 

With both aniline gentian-violet and with carbol-fuchsin, 
methylated spirit should be used to clear for one minute. 

These four stains are of general application, and although by no 
means all that are used by bacteriologists are sufficient for most 
purposes. Various special methods will now be described, and the 
composition of the stains given under the various headings. 

Gram's Method. — Solutions : 

(1) Aniline gentian-violet. 

(2) Iodine, 1 gm. ; pot. iod., 2 gm. ; distilled water, 300 cc. 

(3) Absolute alcohol or rectified spirit. 

(4) Xylol. 

(5) Balsam in xylol. 

Method :— 

(1) Stain the section of tissue or coverslip film for five minutes, 
preferably by floating on the stain in a watch glass. 

(2) Place in iodine solution, after washing off excess of stain, 
till the colour has changed to purple black ; time required one-half 
to two minutes. 

(3) Decolourise in absolute alcohol till no more colour can be 

(4) Wash well in xylol, dry and mount in Canada balsam 
dissolved in xylol. 

All bacteria do not stain by this method, and it is therefore used 
as a means of differentiating certain species. 

There are various modifications of the process ; two only will 
be mentioned, (a) Weigert, who uses aniline oil as the decolourising 
agent after the iodine solution, instead of alcohol, (b) Muir and 
Eitchie, who use carbolic instead of the aniline water (1), and 
substituting olive-oil for the xylol (4), afterwards washing in xylol. 
Contrast stains may be employed with this method, and have the 
advantage of showing any bacteria present that have decolourised 
by the Gram process. 

Safranin and Bismarck brown, saturated alcoholic solution diluted 
with an equal bulk of distilled water, give good results. 

Ziehl-Neelsen method for acid-fast bacteria (tubercle, leprosy, &c). 
The tubercle bacillus does not stain well with the ordinary methods 


adopted for bacteria generally, but requires an energetic stain plus 
the application of heat. 

Solutions : — 

(1) Carbol-fuchsin stain. 

(2) 25 per cent, of pure sulphuric acid. 

(3) Alcohol, absolute or rectified. 

(4) Carbol-niethylene blue stain. 

Method : — 

(1) Stain the films for five or ten minutes in diluted carbol- 
fuchsin (1 in 3 water) kept hot over a water bath. The stain should 
steam but not boil. 

(2) Decolourise with rectified spirit until no more colour is 
extracted, and wash in water. 

(3) Plunge into the sulphuric acid solution and wash in water ; 
repeat the process if there is more than a faint pink tinge on 

(4) Stain for half a minute in diluted carbol-niethylene blue. 
Wash, dry, mount. The tubercle bacilli are stained a bright red, 
the background of epithelial cells and other bacteria, blue. 

Spore Staining. — Solutions : 

(1) Carbol-fuchsin. 

(2) 5 per cent, sulphuric acid. 

Method I. 

(1) Stain the film as for tubercle bacilli. 

(2) Decolourise rapidly in 5 per cent, sulphuric acid and wash. 

(3) Counterstain with carbol-methylene blue. 

(4) Wash, dry, and mount. 

The spores are stained a bright red, the bacilli blue. 
Holler's method of spore-staining is of considerable advantage in 
some cases: — 

Method II, 

(1) Chloroform two minutes. 

(2) 5 per cent, chromic acid two minutes. 
The remaining steps are similar to Method I. 

In all the above preparations the film is prepared as directed on 
page 41. To simply demonstrate the presence of spores without 
attempting to stain the organisms the flaming is increased. This 
makes the spore envelope more permeable to the stain, although the 



bacilli themselves are broken up and easily decolourise in the acid 

Method HI. 

(1) Pass coverslip fifteen times through flame. Proceed as in 
I., but do not counter-stain, as the bacilli are destroyed by the 

Capsule Staining. — Some organisms, as the pneumococcus, are 
possessed of gelatinous capsules, which may be stained by special 

Method I. 

Solutions : Glacial acetic acid ; aniline gentian-violet ; sodium chloride 
2 per cent. 

(1) Immerse in acetic acid while film is wet for three seconds. 

(2) Wash off acid with aniline gentian violet. 

(3) Wash in 2 per cent, sodium chloride. 

(4) Examine in sodium chloride solution. 

Fig. 15. — Boston's Forceps for holding Coverslips during Staining. 

Fxq. 16. Cornet's Forceps for holding Coverslips during Staining. 

MacConkeys : Method II. 

Stain: Dahlia 1'5 gm. 

Methyl-green (00 crystals) . . . . 0-5 gm. 

Sat. alcoholic fuchsin . . . . . . 10*0 cc. 

Distilled water 200 cc. 

(1) Prepare film in ordinary way. 

(2) Flood coverslip with stain holding it in spring forceps 

(3) Heat till steam is given off, and allow to remain five minutes. 

(4) Wash, dry, and mount. 

The cocci are stained a deep violet, the capsules a faint violet. 


Flagella. — Flagella staining is one of the most difficult of all 
bacteriological operations, requiring a good deal of practice and not 
a little patience. The flagella are invisible under ordinary circum- 
stances, and it is probable that in the process of staining they 
become swollen and so come into view. Another idea is that the 
stain is deposited upon and not in the flagella, as is the case in the 
ordinary staining of bacteria. It is most difficult to get both flagella 
and bacilli stained and the beginner must be prepared for a good 
many failures before a successful preparation is obtained. 

In staining flagella the greatest care must be exercised that the 
coverglasses used are perfectly clean, that the films are not too 
thick, that the organisms are well separated in the emulsion used, 
that the flagella are not destroyed in flaming, and that the stains 
used do not precipitate. A young agar culture should be used, and 
a small quantity of the growth removed with the platinum needle 
and emulsified with distilled water in a clean watch glass. A drop 
of this emulsion is carefully spread over the surface of the clean 
coverslip and allowed to dry, and then passed once or twice through 
the flame. Three methods are described, although many others 
have been suggested from time to time. It will be seen that a 
mordant is used in all cases. 

PittfielcVs Flagella Stain. 

Solutions : A. Saturated alcoholic gentiau-violet . . 1 part. 
10 per cent, solution of potash alum. . 10 parts. 
B. 10 per cent, solution of tannic acid 

Mix equal parts of A and B immediately before use. 

Method : Flood the coverslip with the mixture and warm over 
bunsen flame till steam is given off, but do not boil. Allow the 
stain to remain on for five minutes ; wash off, dry, and mount. 

van E r men g em's Flagella Stain. 

A. Osniic acid 2 per cent, solution . . . . 1 ccm. 

Tannin 20 per cent, solution . . . . 2 ccm. 

Acetic acid (glacial) . . . . . . . . 5 drops. 

Mix, and keep till distinct violet colour. 

B. Silver nitrate .. .. .. .. .. 0'5gm. 

.. 100 cc. 

Distilled water 

Gallic acid 


Fused potassium acetate 

Distilled water. . 



10 gm. 

350 cc. 


Method : Place films in solution A for half an hour, wash well 
in distilled water, and then in absolute alcohol. Transfer to solu- 
tion B for twenty seconds, and then without washing place in solu- 
tion C for ten seconds. The film is again placed in solution B till 
it commences to turn black, and then washed, dried, and mounted. 

McCrorie's Flagella Stain. 

Solutions: A. Saturated alcoholic " night-blue ". . 1 part. 

B. 10 per cent, solution potash alum . . 1 part. 

C. 10 per cent, solution of tannin . . 1 part. 

Method : Mix immediately before staining, place film in incubator 
for half an hour. Wash, dry, and mount. The stain works best 
about an hour after mixing. 

Neisser's Stain for Diphtheria. 

A. Methylene blue . . . . . . . . . . 1 gm. 

Absolute alcohol . . . . . . .' . 20 cc. 

Glacial acetic acid . . . . . . . . 50 cc. 

Distilled water . . . . . . . . . 930 cc. 


B. Bismarck Brown . . . . . . . . 2 gm. 

Distilled water 1,000 cc. 

Stain one minute in A, wash, and stain one minute in B. 

Fig. 17. — Bacillus typhi abdominalis flagella stained by 

Pitfield's process. 

x 1,000. 

From Washbourn and Goodall's Infectious Diseases. 



Methods of Observing Bacteria— Cultivations — 
Culture Media. 

Methods of Observing Bacteria — Cultural. 

In the cultivation or growth of bacteria in the laboratory various 
solutions and jellies are made use of, some of which are adapted 
to the development of the majority of micro-organisms, others 
adapted to certain species. These culture media contain the sub- 
stances peculiarly suited to the growth of bacteria, while at the same 
time they are easy to manipulate. 

The largest number of bacteria which are to be found in the 
mouth will grow perfectly well upon the commonly used laboratory 
media ; notwithstanding this a considerable number of observers 
have adopted various special varieties of media with which to carry 
on their labours, without at the same time giving details of the 
growth upon the media common to all proper bacteriological work. 
In this way it is often most difficult to determine the exact relation- 
ship of many bacteria which have been described, often most 
imperfectly, as occurring in the mouth. Certain mouth bacteria 
no doubt require special media for their isolation, but once isolated 
they will generally develop on the usual laboratory test media. It 
is clearly essential therefore that the same methods and composition 
of media should be adopted in all descriptive- work of new bacteria 
in order that real progress may be made ; bacteria as a rule are so 
influenced by their environment that the greatest care has to be 
exercised in making the standard media. 

I shall therefore give the details of ordinary laboratory media 
making in full, as adopted in the majority of bacteriological labora- 
tories both in this country and on the Continent. The formulae for 
special media are also given, the detail where omitted is similar 
to that described for the preparation of ordinary media. The 



reader will have already gathered from the chapter on morphology 
that many species of bacteria have the same morphological form 
as determined by microscopical examination, and therefore the 
method of cultivation is invoked to establish easily recognised 
differences between the various species. 

Fig. 18.- 

-Appabatus arranged for filling Test Tubes with 
Nutrient Solutions. 

The glass three-way tap is connected to the reservoir funnel with a rubber 
tube. The liquid is allowed to flow from the funnel into the glass measuring 
cylinder at the top of the three-way tap till it reaches the 10 cc. mark ; the 
tap is then turned sharply and the fluid flows into the test tube. 

Culture media may be either fluid or solid, natural or artificial, 
the majority having a common basis of watery extract of meat ; 
this will be described first. 


Nutrient Broth or bouillon forms one of the most useful media, 
and moreover the basis of many of the jellies or solid media used. 

Preparation. — A pound of lean beefsteak is finely chopped up 
and passed through a mincing machine (the fat and connective 
tissue are removed first). A litre of distilled water is added and the 
whole digested for half an hour at 6(P C. ; the mixture should be 
kept constantly stirred. 

The temperature is now allowed to rise to 100° C. and the 
fluid filtered off through filter paper, and the filtrate made up 
to the litre with more water. To the filtrate 1 per cent, of peptone 
(10 gm.) and Oo per cent. (5 gm.) of sodium chloride is added. 
The peptone and salt are best mixed up with 25 cc. of the broth 
into a thick emulsion and then added to the bulk, boiled for 
half an hour, neutralised and filtered. The filtrate is nutrient 
broth. In preparing agar and gelatin, the agar or gelatin is added 
at the same time as the peptone. 

Fig. 19. — Erlenmeyer Flask. 

In neutralizing the broth two methods maybe adopted, the point 
aimed at however should be to use a method which shall give con- 
stant results so that the finished media shall be of a definite alkalinity 
or acidity expressed in terms of standard alkali, otherwise successive 
brews will not have a constant definite reaction. Bacteria, as has 
been pointed out, are extremely sensitive to their environment, and 
the differences of alkalinity in two batches of media modify the 
cultural characters of many bacteria to a surprising extent. Con- 
siderable care must be exercised therefore in neutralizing media. 

Phenolphthalein Method. — Eeagents, &c, 5 per cent, of phenol- 


phthalein in 50 per cent, alcohol. Burette graduated in tenths of a 
cubic centimetre. 25 ccm. pipette. Boiling distilled water in a 
wash bottle. Normal and decinormal sodium hydrate. Beakers. 

Method.— Fill up burette with ^ NaOH (10 cc. = 1 cc. f NaOH). 
Withdraw 25 cc. of the broth (agar or gelatin) and run into the 
beaker, wash out the pipette with boiling distilled water into beaker, 
keep contents at boiling point. Add 3 or 4 drops of the phenol- 
phthalein solution. 

Eead burette and carefully run in the T ^ NaOH until a faint flesh 
colour appears. Eead burette. Make a control and adopt the mean. 

The titration must be made at boiling to eliminate C0 2 , which 
will interfere with the reaction. 

The number of cubic centimetres of decinormal sodium hydrate 
used (~q NaOH) will give the number of cc. required to render 25 cc. 
of broth neutral to phenolphthalein. 

Example — 
Suppose that 25 cc. required 6-0 cc. of -^o NaOH to produce a faint pink colour. 
25 cc. ,, 6-25 cc. 
25 cc. „ 6-05 cc. 

Average for three estimations 6*1 cc. -^o NaOH. 
Therefore — 

100 cc. will require 6 - l x 4, 
And 1,000 cc. „ „ 6-1 x 4 x 10 = 244 cc. JL NaOH = 24-4 cc. J NaOH. 

But we have removed 75 cc. for estimation, therefore we have 
left 1000 -75 = 925 cc. It follows therefore that 925 cc. will 
require 22-6 cc. £ NaOH to render it neutral to phenolphthalein. 

Only a few organisms will show maximum development in a 
medium with such a reaction, but it has been found by various 
workers, especially Eyre, that the reaction for the majority of 
bacteria is at an optimum when the medium still requires 10 cc. 
~ NaOH to be added per litre to bring the reaction up to neutral 
to phenolphthalein. The medium which still requires this 10 cc. 
J NaOH per litre is said to have a reaction of "plus 10," or 
10 degrees of acidity to phenolphthalein. 

In the example above, then, it is necessary to deduct 9'25 from 
the estimated quantity for absolute neutralization (22-5 cc), and 
the addition therefore of 124 cc. J NaOH to our broth will give 
it a reaction of -f 10. Other degrees are expressed as indicated 
+ 5, + 4, + 8, and so on. 



By this means it is always possible to prepare a medium of 
" standard reaction." 

Litmus Method. — The hot medium (broth, agar, or gelatin) is 
gradually neutralized by dropping in J NaOH from a burette and 
testing the reaction on litmus paper. When the medium is neutral 
to litmus 4-5 cc. more of J XaOH are added to each litre. This 
method is much quicker, but does not take into account the 
NaH 2 PO i , and is therefore uncertain, especially as many weak 
organic acids do not react to litmus at all well. 

Fig. 20.— Hot-water Funnel. 

Note. — In making up the filtrate to the litre when hot the method 
adopted is to weigh the medium in a tared flask and add the requisite 
amount by weight, having weighed the whole at the commencement. 

(<rt) Agar (ordinary nutrient). — Powdered agar-agar, 2 gms. ; 
nutrient broth, 100 cc. 

The agar powder is mixed to a thin cream with some of the broth 
and added to the bulk. The mixture is then boiled in the steam 
sterilizer until all the agar is dissolved, neutralized while hot in 
the manner described for broth, and cooled to 60° C. The white 
of two eggs beaten up in distilled water are added, and the materia 
boiled in the steamer until the whole of the flocculent precipitate 


has fallen, which generally takes three-quarters to one and a quarter 

It is now ready for filtering ; the ordinary filter papers are too 
fine for the purpose, and the ones generally used are the thick white 
" Chardin " filters. The filtering may be carried out in a hot water 
funnel (fig. 20), or in the steam sterilizer, and is a somewhat tedious 
process. When filtered the clear filtrate is passed into test tubes 
and sterilized by the intermittent method. 

To avoid filtering the medium may be boiled, after the egg is 
added, in tall beakers and allowed to cool therein ; the flocculent 
precipitate falls to the bottom and may be removed with a knife 
when the mass has solidified. The agar made in this way is not so 
clear as when filtered. 

Some bacteriologists boil their agar in the autoclave, but the 
great objection to this is that the medium tends to turn brown. 

(b) Glycerine Agar. — This medium, first introduced for the cul- 
ture of the tubercle bacillus by Eoux and Yersin, is ordinary nutrient 
agar to which 10 per cent, of glycerine has been added. The 
preparation is as (a). 

(c) Glucose Formate Agar. — This medium, which is largely used 
in the cultivation of anaerobic organisms, is ordinary nutrient agar 
to which 2 per cent, of glucose and 0'5 per cent, of sodium formate 
have been added. Kitasatio, who introduced the method, did so 
on purely theoretical grounds, the elaboration of proteid taking place 
in its primary synthesis by the formation of aldehyde (Hueppe). 

(d) Agar Streaked with Blood. — This medium is used in the 
cultivation of the pneumococcus, and gonococcus particularly. 
Fresh human blood may be used, or that of an animal; in either 
case great care must be exercised in excluding adventitious bacteria. 
The method generally adopted is to take a rabbit, wash the ear 
well with lysol and soft soap and shave off the hair. Again wash 
with lysol and finally with alcohol. The large vein is then punc- 
tured, and the escaping blood removed with sterile pipettes and 
smeared over the surface of slanted agar tubes. The tubes prepared 
in this way are incubated for twenty-four to forty-eight hours in 
the hot incubator at 37 - 5° C, and if no development of colonies 
takes place are ready for use. 

Various other substances are added to agar for special purposes ; 
the basis in all cases is the ordinary nutrient agar (a). The follow- 
ing are some of the varieties : — 



(e) Iron Agar. — 2 per cent, of saccharate or tartrate of iron. 

(/) Sugar Agar. —~SIa\tose and lactose, 5 per cent., &c. 

(g) Gelatin Agar.— Broth, 100 cc. ; agar, 15 gm. ; gelatin, 
7*0 gm. Prepared as agar (a). 

(h) Gelatin.— Ordinary nutrient gelatin is a medium largely em- 
ployed. Its composition is : best French gelatin, 10 gm. ; nutrient 
broth, 100 cc. 

The gelatin is dissolved in the broth by heat, neutralized while 
hot (as above), cooled to 60 : C, the white of an egg added, boiled 
for half an hour, filtered in a hot water funnel and run into sterile 
tubes, and sterilized by intermittent method. 

The sterilization of gelatin in the autoclave generally results in 
the peptonisation of the gelatin, in which condition it will not set 
on cooling ; 20 per cent, gelatin is also employed at times. 

Pig. 21. — Potato Cutter. 

Fig. 22. — Roux's Potato Tube, the lower end arranged to catch condensa- 
tion water. 

(/) Glycerine Gelatin. — Gelatin, 10 gm. ; glycerine, 4 ccm. ; 
broth, 100 ccm. Prepare as gelatin. 

(j) Glycerine may also be added to ordinary broth in the same 
proportion (4 per cent.). 

(k) Potato. — A good sized potato is well washed with a brush 
and hot water, peeled, and the eyes removed. With a circular potato 
cutter (fig. 21) cut out cylinders about four inches long, and wash 
well with water. Divide the cylinders longitudinally so that each 
has a broad and narrow end. Drop them into sterile Roux's tubes 
or tubes with small plugs of wool at the bottom and sterilize in the 
steamer. If the potatoes are acid wash the cut slices in 2 per cent, 
caustic soda for an hour before placing in tubes. Glycerinated 
potato is also used. The slices are soaked in 6 per cent, solution 
of glycerine in water before they are placed in the tubes. 



(I) Potato Gelatin. — Peel several potatoes and remove the eyes, 
weigh out 1 kilo., cut up in mincing machine, and add 1,000 cc. of 
water. Allow to stand twenty-four hours. Filter. Add 1 per cent, 
asparagin and 4 per cent, glycerine, 10 per cent, gelatin, and the 
white of an egg. Boil up, filter, and run into tubes. 

Potato water is made in a similar way, but without gelatin. 

(m) Neutral Litmus. — Two ounces of commercial litmus are 
extracted with rectified spirit for thirty days, changing the spirit 
three times. At the end of this time the litmus is emptied into a 
flask, and the spirit allowed to evaporate ; 600 cc. of filtered water 
are next added, the litmus dissolved up and filtered, and acidified 
with pure sulphuric acid. An excess of barium hydrate is then 
added, and the solution again filtered. Carbon dioxide is then 
passed through till all the barium is precipitated as carbonate, the 
solution filtered and sterilized. 

Sufficient of this solution to give a good blue colour is added to 
the various media when required. 

(n) Litmus Milk. — 500 cc. of milk are run into a funnel and 
heated in the sterilizer, and allowed to stand for twenty-four hours 
till the cream has risen. The milk is then drawn off, tinted by 
addition of neutral litmus, run into tubes and sterilized in the 

(o) Blood Serum. — The blood of an animal is collected in a large 
sterilized jar at the slaughter house ; the first runnings are allowed 
to escape to avoid contamination from the skin, &c. The jar is 
then filled and the blood allowed to clot. The serum is pipetted off 
with a sterile pipette and run into tubes, which are placed in the 
inspissator in a slanting position, and sterilized by heating to a 
temperature of 75° C. for half an hour on four or five successive 
days. Eeject any tubes that show growth when incubated after 
the last sterilization. 

(p) Peptone Water (Durham's). — Water 100 cc. ; peptone 1 gm. ; 
salt 0*5 gm. Boil for twenty minutes, filter and run into tubes and 
sterilize in the usual manner. Instead of 1 per cent, more peptone 
may be added up to 4 per cent. 

Peptone water is a useful medium to use in determining the 
fermentation of carbohydrates. Various amounts may be added, 
2 per cent, being an average quantity. The solution may be also 
coloured with neutral litmus. Glucose, lactose, maltose, starch, &c, 
may be used. 


(q) Beer Wort Gelatin, and Agar. — Beer wort is used instead of 
broth in making these media ; it is best not to neutralize. 

(r) Nitrate Media. — 0*5 per cent, of potassium or sodium nitrate 
may be added to test the reducing power of organisms upon nitrates. 
Broth, gelatin or agar may be used. 

(s) Bread is a good medium for moulds. Dry bread is grated and 
the crumbs placed in small Erlenmeyer flasks and just covered with 
distilled water. The flasks are sterilized on four succeeding days 
in the steamer. 

(t) Media with Iron Salts. — 2 per cent, of iron lactate or 
saccharate may be added to agar, gelatin or broth, for testing the 
production of sulphuretted hydrogen. 

(u) Inosit Free Broth. — Bacillus coli is grown in the beef extract 
before addition of peptone, Sec. After eighteen hours at 37° C. the 
flask is placed in the steamer for an hour and the contents sub- 
sequently used for making agar, gelatin or broth. The organisms 
use up the muscle sugar (inosit). 

(v) Saliva Media. — Saliva is obtained by placing a sterile Woolfe 
bottle on the draining tube of a saliva ejector whilst the latter is in 
use for dental operations. The saliva is boiled, 1 per cent, peptone 
added, filtered and sterilized in the tubes. Gelatin 10 per cent, or 
agar 2 per cent, may be added for solid media. The saliva should 
be sterilized as soon after collection as possible ; if not treated in 
four hours it must be rejected. For some operations the fresh 
saliva is filtered through a Pasteur-Chamberland filter. (Figs. 11 
and 30.) 

Methods of Cultivating Bacteria. 

Inoculation of Culture Tubes. — The operation of planting a 
substance containing organisms on to nutrient media for the pur- 
pose of obtaining a culture is termed inoculation, and is performed 
with a platinum wire mounted in a glass, or better, aluminium 
handle. Two wires are required : (a) a straight wire slightly flattened 
at the end into a spatula ; (b) a wire terminating in a loop — two of 
these, different sizes, are useful (fig. 23). Before and after use the 
wire is heated to redness in the flame, before use to burn off any 
adhering organisms that would contaminate the culture, afterwards 
to remove the organisms taken from the tube and still remaining 
attached to the wire. 

To inoculate one tube from another containing a cultivation 


the following method is adopted : — Flame, i.e., set fire to the cotton 
wool plugs of the two tubes to burn off any dust (bacteria) which 
may have fallen on to the stopper, then blow out the flame. Hold the 
tubes together in the left hand between the thumb and first two 
fingers, take up the platinum wire like a pencil and sterilize it in 
the flame ; with the little finger of the right hand remove the cotton 

Fig. 23. — Platino-iridium Inoculating Needles, Loop and Spatula. 

wool plug from one of the two tubes, keeping them inclined at an 
angle to avoid spores, &c, dropping in; remove the second plug with 
the third finger, holding the two plugs in the right hand (see fig. 24). 
Flame the open mouths of the two tubes and then remove a small 
portion of the culture from one tube and transfer it to the other, 

Fig. 24. — Method op Inoculating one Tube prom Another (viewed from 
above). Note the method of holding the cotton-wool plugs. 

carefully avoiding touching the edge or sides of the tubes in the 
process ; replace the plugs, flame the wire, and then the tube plugs 
again, label and place the inoculated tubes in the incubator. Exactly 
the same procedure is adopted if the cultivation is made from any 
material from which we wish to cultivate bacteria ; when the 
material is fluid the loop is used. 



Solid media are inoculated in three ways: (a) "streak," (b) 
" stab," (c) " shake." 

Streak Cultures. — The media is first " sloped," i.e., melted, and 
the tube laid down at an angle, care being taken that the fluid does 
not touch the plug ; several tubes should be sloped at once, and a 
folded duster placed over them while they cool, to prevent dimming 

Fig. 25. — Hearson's Incubator. 

The apparatus consists of a copper tank filled with water enclosed in a 
wooden frame. The temperature is maintained by a gas jet, the supply to 
which is regulated by a capsule with a definite boiling point. The excursions of 
the capsule are communicated to a valve which automatically regulates the gas 
supply. Two incubators are required, one working at 37 "5° C, the other at 22° C. 

by condensation. When sloped the tubes should be kept twenty- 
four hours before using. With agar it is a good plan to add a little 
gum arabic or gelatin to the media to prevent the sloped media 
slipping to the bottom of the tube. To inoculate the sloped surface 



draw the platinum needle or loop, charged with the culture, gently 
up the surface of the medium and replace the plug. The conden- 
sation water in agar tubes should not be poured out, as it has a 
characteristic appearance with certain organisms. 

Stab Cultures. — These, like the latter, may be made on agar 
or gelatin ; the tubes are not sloped. A charged needle is passed to 
the bottom of the medium and withdrawn. This method is largely 
used in making cultures of anaerobic bacteria and determining the 
production of liquefaction in gelatin. 

Shake Cultures may be used to determine anaerobiosis, production 
of gas, &c. The solid medium is melted in a water bath, con- 
veniently fitted with a rack perforated to allow of the tubes 
standing upright; when thoroughly melted the medium is allowed 
to cool to 40° C, inoculated as described above, and then well 
shaken by four or five rapid swings (not up and down). When 
set the tube is placed in the incubator. 


Fig. 26. — Enlaeged View op Gas Valve in Heaeson's Biological Incubatoe. 

A, Gas supply ; B, tambour ; C, gas supply to jet ; D, weight on lever to 
regulate size of flame ; P, pin communicating with capsule in the incubator ; 
S, sideway tap. 

Liquid Media. — The loop or spatula charged with material 
should not be directly inoculated into the fluid, but the inside of 
the tube just above the meniscus is touched with the charged wire ; 
a loopful of the medium is then taken up and mixed with the drop 
of material so that a complete emulsion is formed. On placing the 
tube upright and giving it one or two swings the material is diffused 
through the tube. 

Plate Cultivations. — Bacteria rarely exist in nature alone and 
are invariably associated with other species, it is therefore necessary 
for the bacteriologist to adopt a method of separating the various 


■species. This is usually done by means of plates The procedure 
is the same for gelatin and agar. 

Three tubes of agar or gelatin are melted in the water bath and 
cooled to 40° C, and one tube inoculated with the mixed culture and 
shaken up. Two loopfuls of this tube are now transferred to a 
second tube and four or five loops of the second into the third. 
The plug of each tube is removed and the contents poured into a 
sterile Petri dish, which has been placed upon the levelling tripod, 
the glass dish of which has been filled with warm water at 40° C. 
The mouth of the tubes are flamed before pouring out the contents, 
and the lip of the tube used to assist the medium to flow over the 
dish. After the plates have set they are incubated. In summer it 
is often necessary to put ice into the reservoir water, otherwise the 
gelatin does not set for hours. 

The amount of media placed in the first tube determines the 
number of loopfuls used for the subsequent dilutions, in fact the 
whole process can only be satisfactorily learned by practice in the 

In determining the number of bacteria present in a sample of, 
say, drinking water, a sterile pipette graduated in tenths of a cubic 
centimetre is used, and 0*5, 03 and Ol ccm. of the water added to 
the various tubes, which are then plated. 

In the process of plating the point aimed at is to separate the 
bacteria from one another in such a manner that when they develop 
in the nutrient substratum the colonies each one forms are suffi- 
ciently separated to observe and make sub-cultivations from, or in 
the water-dilution plates to count the individual colonies, each colony 
representing one organism in the original sample, the total number 
of colonies on the three plates representing the number of bacteria 
present in a cubic centimetre of the water examined. A sub-culture 
made from one of these colonies will generally be found to be pure, 
i.e., will consist of one species of organism alone. 

Having obtained cultivations from a plate colony the culture is 
examined by means of the hanging drop and coverslip preparation 
stained in the various methods given above, and then sub-cultured 
into the various test media, coverslip preparations of each being 

The test media to be used should always include the following : — 
gelatin stab, streak, shake, plates, agar streak, broth, litmus milk, 
blood serum, potato, media containing carbo-hydrate, and others 
containing nitrate. 


So far I have only described the cultivations of aerobic organisms, 
the anaerobic bacteria requiring special methods. 

Glucose formate media are especially adapted to the growth of 
anaerobes, but other media may be used. 

The oxygen of the air must be excluded by one of the following 
methods : — 

(a) Buchner's Tubes (fig. 27). — A large boiling tube fitted with an 
india-rubber cork is used, and a little pyrogallic acid placed in the 

Fig. 27. — Buchner's Tube for Anaerobic Cultivations, with 
Culture Tube in Situ. 

bottom ; some caustic soda solution is then added, the culture tube, 
previously inoculated, gently dropped in and the rubber cork 
replaced. This method is adapted for fluid or solid media. 

(b) An agar or gelatin stab is made in a tube containing about 
twice as much of the medium as is used for ordinary purposes, and 
the top of the stab covered by pouring in a little agar or gelatin 
which has been melted in another tube. 

(c) Hydrogen or other indifferent gas (p. 17) is used to drive out 



the air. The culture may be placed in a special jar with two tubes 
leading into it (fig. 28). Along one is passed hydrogen washed by 
passage through (a) 10 per cent, solution of lead acetate, (b) 10 per 
cent, solution of silver nitrate, (c) 10 per cent, solution of pyrogallic 

Fig. 28. — Bullock's Anaerobic Apparatus for Hydrogen and Alkaline 
Pyrogallic Acid. 

Fig. 29.— Wolff Bottle for Use with Bullock's Apparatus. 
Three are used : (a) lead acetate solution ; (b) silver nitrate solution ; (c) pyro- 
gallic acid and soda. 

acid in caustic soda. Both inlet and outlet tubes are provided 
with cocks which are closed when the gas has been passed till no 
air remains. The lead solution removes any H 2 S, the silver solution 
any CI, and the pyrogallic acid traces of oxygen. The air should 
also be filtered through cotton wool to remove any bacteria. 

Sloped tubes with a tightly-fitting indiarubber cork may be 


used in 'the same way, the two tubes being sealed in the name 
when the gas has been passed for niue or ten minutes. 

VignaVs Method is often a useful one. A piece of thin glass 
tube is heated till it is sterile and either end drawn out. The 
inoculated and melted medium, glucose formate agar for instance, 
is then drawn up into the tube until it is quite full, when 
the ends are sealed by fusing in the flame. It is best to pass hydro- 
gen through the tube beforehand. To obtain sub-cultures the tube 
is marked with a file opposite a colony and the tube broken across. 

Anaerobic cultures in fluid media are also made by using an 
ordinary filter flask and passing hydrogen over in the manner 
described above. 

If, as is often the case, we wish to collect and examine the gas 
produced by anaerobes, a flask, a bent tube — the end of which dips 
into a trough of mercury — is used and the gas collected by dis- 

The Study of Cultures. 

In order to properly recognise a given organism it is necessary 
to make use of the culture media whose composition and mode of 
preparation has been considered. 1 Some organisms, it is true, have 
typical and well-marked characters upon certain media, and an 
experienced eye can therefore pick out some of the well-known 
forms ; but for all general purposes of recognition and differentia- 
tion the appearances and chemical reactions on a number of different 
media must be employed (for chart see page 69). The study of 
cultivations, therefore, gives the mycologist his methods of detecting 
and isolating any particular bacterium, and it is therefore of the 
greatest importance that some general scheme be adopted, both for 
the convenience of the individual worker and for the interests of the 
science at large. 

The descriptions of cultural characters given by many workers 
are often unintentionally misleading from the unavoidable fact that 
many organisms do not conform to a rigid rule, and moreover from 
the avoidable confusion of not using standard media. 

To facilitate the general description of bacteria, Chester has 
recently suggested the introduction of the general botanical nomen- 
clature in the description of cultural characteristics, the terms used 
indicating general types rather than minute and particular descrip- 

1 See page 51. 


tions of isolated examples. Where possible I have placed these 
terms in brackets ; the descriptive terms suggested by Chester, with 
diagrams of the various forms to which the terms apply, are given 
in the appendix. 

The colonies formed by bacteria upon plates of nutrient media 
should be carefully examined both with the naked eye and with a 
§ objective, many bacteria producing typical forms on the various 
media. Both deep and surface colonies should be examined, the 
colonies in the deep layers often appearing to differ widely from 
those growing on the surface, even in pure cultivations. 

On the sloped surface of agar, gelatin, blood serum, potato, or 
other solid medium the general characters of the growth, the contour 
of the edge, the surface, its consistency, &c, are to be noted, as well 
as changes in the medium, such as liquefaction and pigmeutation. 

When a pigment is formed the solubility should be tested in 
various solvents, and, if possible, the spectrum noted. In stab 
cultures several other points are generally noted — the special form in 
which liquefaction of gelatin occurs, the formation of gas and the 
presence of growth along the track of the inoculation needle, the 
character of the surface of the growth at the point of entry of the 
needle, &c. (See diagrams in appendix.) 

In fluid media, besides the general character of the growth, note 
pellicle, or precipitate, turbidity, gas production and acid production, 
as by the alteration in the tint of litmus added to the solution 
various chemical tests may also be applied. 

In broth cultures various other tests may be applied foi 
ammonia, nitrates, sulphuretted hydrogen, &c. 

Peptone water, or broth cultures, with the addition of 1 to 2 
per cent, of various carbo-hydrates, are used for the determina- 
tion of fermentation indol and acid production. For gas formation 
Durham's tube is most useful. A small test tube is placed in the 
culture and sterilized with the medium ; any gas formed collects in 
the floating tube, displacing the contained fluid. 

Theobald Smith's tube may be also used. The bulb is filled 
with glucose-broth and the gas formed collects in the large end of 
the tube, from which it may be removed for analysis. The amount 
of C0 2 may be determined by filling the bulb with NaOH and 
shaking up ; the loss in volume represents the C0 2 absorbed. 

Proteolytic enzymes may be also tested for in broth cultures 
as follows : — A broth culture of known liquefying organism is 


grown for four or five days, and at the end of this time some of the 
contents are poured into a tube of thymol gelatin and a small piece 
of thymol added. A control tube is filled with water and thymol 
added. If any enzyme is present liquefaction takes place in the 
tube containing the culture, the organisms being prevented from 
developing by the thymol. 

The enzyme may be isolated if desired by extracting with thymol 
water, precipitating with absolute alcohol, and taking up precipi- 
tate in thymol water again. Certain sugars which do not reduce 
Fehling's solution are inverted by bacterial activity into reducing 
sugars ; these are tested for with Fehling in the ordinary way. 

Fig. 30.— Filter Flask with Pasteur-Chamberland Filter ready for 
Filtering Toxine. The rubber tube is connected with an exhaust pump. 

Alkali albumin in broth and ordinary nutrient broth are used to 
obtain the toxins of certain bacteria, such as diphtheria. The 
organisms are grown for seven days at 37° C, and then filtered off 
by means of a porcelain filter candle (fig. 30) ; the filtrate con- 
taining the toxins may be tested by injection into animals. 

For the isolation and quantitative determination of the various 
organic acids, &c, produced by bacteria from carbo-hydrates, 
fractional distillation must be employed, details of which are given 
in appendix. 

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Notwithstanding the statements of a certain small class of 
unscientific persons the experimental inoculation of animals has 
undoubtedly led to great advances in medicine and surgery redound- 
ing with benefit to the human race, and has placed many new and 
valuable facts of treatment and diagnosis within the reach of those 
engaged in alleviating suffering and combating disease. 

The animals chiefly used in experimental inoculation by bac- 
teriologists licensed to perform such experiments are rabbits, guinea 
pigs, rats and mice, whilst in the production of antitoxine on 
a large scale horses are generally employed. The experimental 
inoculation may be made in various ways. 

(a) Subcutaneous Injection, the material used being injected 
under the skin on the abdominal surface in guinea pigs and rabbits, 
and at the root of the tail in mice. Solid matter is thus introduced. 

(b) Intraperitoneal Inoculation. — The fluid is introduced into 
the peritoneal cavity direct by means of a sterilised hypodermic 

(c) Intra-venous Inoculation, the injection being made directly 
into the veins, generally the posterior vein of the rabbit's ear. 

(d) Intra-muscular Inoculation. — The injection is made deeply 
into the muscles. Various other methods are also employed, such 
as mixing the infecting material with the animal's food, intercranial 
injection by removing a portion of the skull with a small trephine ; 
inoculation into the anterior chamber of the eye, &c. The animal 
is placed in a separate cage after inoculation and watched, the 
symptoms noted, and if death occurs a careful autopsy made. To 
avoid any contamination sterile instruments (well boiled) are used, 
and the surfaces of glands, heart, &c, well seared with a hot 
iron before cultivations are made. Cultivations and coverslip 
preparations are made from the site of inoculation, peritoneal fluid, 
heart blood and spleen, and from any other sites that the special 
case under consideration suggests. 

For the full particulars of experimental inoculation the reader is 
referred to the laboratory text-book by Eyre ;* and it must be remem- 
bered that, owing to the stringent regulations in force, no one may 
perform inoculation experiments under pain of heavy penalty unless 
they hold a licence from the Home Secretary. 

' Bacterological Technique." 


Susceptibility and Immunity. 


General. — By immunity is meant the non-susceptibility to a 
given disease or to a given micro-organism. Such a resistance may 
be natural to a whole genus of animals or may be acquired by an 
animal as the outcome of experimeutal inoculation ; immunity is 
therefore divided into two great divisions — natural immunity as 
possessed by certain animals naturally, and acquired immunity 
developed either as the result of passing through an attack of the 
disease, or as the result of experimental inoculation with the agents 
of the disease (bacteria or their toxines). All diseases do not 
produce a corresponding immunity, whilst others apparently confer 
a very large degree of protection. For instance in the case of small- 
pox, scarlet fever and typhoid fever (enteric), one attack generally 
produces protection lasting many years. On the other hand influenza, 
pneumonia and erysipelas may occur several times in the same 
individual, but even with these a certain transitory degree of 
immunity may be produced. All individuals do not exhibit the 
same degree of susceptibility to a given disease, as is seen in the 
percentages of those developing a disease after exposure to infection 
(c/. diphtheria). 

The immunity shown by various races of man differs for certain 
diseases, and many diseases affecting man are unknown in the 
lower animals and vice versa. 

As a general rule the greater the virulence of the organism 
producing a disease the more pronounced the protection on recovery ; 
in the experimental production of immunity therefore organisms of 
a high degree of virulence are employed in the final inoculations. 

Changes in the Animal organism associated with Immunity. — In 
acquired immunity, and to a small extent in natural immunity, the 


blood of the immunized animal is found to contain certain bodies 
which were not present before the disease was contracted or the 
special inoculations commenced, and the development of immunity 
is coincident with their appearance in the blood. These anti-bodies 
(antikorper) may be of two varieties : (a) anti-toxic, which neutralize 
the toxines produced by the invading bacteria ; (b) anti-bacterial, 
which attack and produce solution of the bacteria themselves. 
These anti-bodies are not always produced in equal quantities and 
vary within wide limits. The antitoxines used therapeutically as 
in treatment of diphtheria, are "antitoxic" rather than "anti- 
bacterial." The formation of anti-bodies is not limited to the 
infection by bacteria by their products, for instance the injection of 
the washed red blood corpuscles of one animal into another results 
in the formation of "hemolysins" which produce solution of the 
red corpuscles of the blood injected, and so on with various other 
substances. Rennet ferment on injection produces " anti-rennin," 
which prevents rennet ferment from acting upon milk. Besides the 
production of these anti-bodies "agglutinins " are formed which cause 
the agglomeration of the bacteria against which the animal has 
been immunised. These bodies are referred to in greater detail 

Artificial Immunity. — Two varieties are recognised : (a) Active 
immunity; (b) Passive immunity (protective). 

Active immunity is obtained by injecting an animal with a non- 
fatal dose of a given bacterium, the dose being so arranged that 
a considerable illness (reaction) with recovery ensues. After an 
interval the injection is repeated with a slight increase in the dose, 
and so on until the animal can withstand many times the initial 
fatal dose (determined by the injection of a control animal of the 
same size and weight) without reaction. The animal is now said 
to be "immunized." Instead of the bacteria themselves, their pro- 
ducts, obtained by filtering broth cultures through porcelain filters, 
may be used, the animal developing immunity as the result of the 
injection of the toxines in the filtrate. 

Active immunity may be produced : — 

(A) By the Injection of Living Organisms Attenuated in 
Various Ways. 
(1) By growing in the presence of air or oxygen. All patho- 
genic organisms gradually lose their initial virulence 
when cultivated for some time outside the body. 


(2) By the inoculation of another species of animal. Thus 

passing anthrax through a guinea-pig lessens its 
virulence for cattle. 

(3) By cultivating the organisms at abnormal temperatures. 

Pasteur found that anthrax was so much attenuated 
that it no longer produced fatal illness in sheep, if 
the cultures were exposed for a certain time to a 
temperature of 55° C. 

(4) By growing the organisms in ivcak antiseptic solutions, 

and sometimes by injecting such solutions with the 

(5) By injection of non-fatal doses of virulent organisms. 

(B) By the Injection of Dead Organisms. 

(C) By the Injection of Filtered Bacterial Cultures. 

(D) By Feeding with Dead Cultures of Bacteria. 
Sometimes it is necessary to increase the initial virulence of a 

given organism. 

Exaltation of Virulence. 

(A) By the metJiod of "passage," first described by Pasteur. 

An animal is injected with an organism either intra- 
venously or intrapcritoneally, and another animal in- 
jected with the blood of the first, containing the 
organisms ; or a culture may be made upon each occa- 
sion. By this means the virulence of the organism may 
be raised to an enormously high pitch. 

(B) By the combined injection with other organisms. 

Thus an attenuated diphtheria bacillus may be raised in 
virulence by injecting it into an animal together with the 
streptococcus pyogenes ; an attenuated streptococcus by 
adding bacillus coli, &c. 

(C) By artificially lowering an animal's resistance by the action 

of heat, cold or overwork, &c, or by exposure to general 
depressing conditions, i.e., guinea-pigs exposed to sewer 
air succumb to smaller doses of diphtheria than do 
control animals. 
By keeping a frog at a temperature of 30° C. it is rendered 
susceptible to anthrax and by cooling the ordinary fowl 
by iced water it becomes less resistant to the cholera 


By neutralizing the stomach contents of an animal im- 
mune to cholera and paralyzing the peristaltic action of 
the intestines with opium Pasteur found choleraic 
symptoms were produced on feeding with the organisms. 

Passive Immunity is that form of resistance to a given disease 
conferred on a susceptible animal by injecting it with the serum 
of an immune animal. 

The immune serum may be inoculated with the cultivation to 
be tested, or subsequently within certain time limits which differ 
for the different organisms used. 

For instance, an animal A is injected with increasing doses of 
diphtheria toxine until it becomes immune. The animal is now 
bled under aseptic precautions and the clear serum separated from 
the clot. 

An animal B, which has not undergone the process of immuniza- 
tion is injected with a fatal dose (determined by previous injection 
of other animals) of the diphtheria toxine, and at the same time with 
the serum obtained from A, B is protected and recovers from what 
would prove a fatal issue without the protection afforded by the 
serum of A. Such serum is termed antitoxic, as it apparently 
neutralizes the toxine. If the animal A is immunized by the injec- 
tion of bacteria instead of their toxines the serum will protect against 
living organisms, that is, the immune serum is antibacterial. 

Passive Immunity may therefore be divided into varieties. 
(^4) Antitoxic — when the anti-serum neutralizes the toxine. 
(B) Antibacterial — when the anti-serum destroys the bacteria 

It follows, therefore, that an antitoxic serum is obtained from 
an animal immunized by toxine injections, and an antibacterial 
serum from an animal immunized by the injection of living bacteria. 
The injection of killed cultures also produces an antibacterial serum 
containing lysins. The serum of an animal immunized by injec- 
tion of living bacteria which produce a toxine may be both antitoxic 
and antibacterial, the two phenomena depending on different 

The production of antitoxine in immunized animals has been 
applied to the treatment of various diseases of a toxic nature, 
especially diphtheria. The antibacterial sera have so far not met 
with any great success in the treatment of disease, although 
attempts have been recently made to protect man against enteric 


fever by direct inoculation of killed typhoid cultures. This method, 
however, does not come under the head of anti-bacterial serum, 
the protection being produced by active immunization. The anti- 
bacterial bodies are important in the diagnosis of certain diseases, 
and will be referred to subsequently. 

Antitoxine. Antitoxic Sera. — The various steps in the produc- 
tion of antitoxine are as follows, and diphtheria may be taken to 
serve as the type of the general method adopted : — 

(1) Preparation of a powerful toxine by growing the organism 
elaborating the toxine under the most favourable conditions for 
the development of the poison. Filtration of the fluid containing 
the toxine through a porcelain filter (see fig. 30), the filtrate being 
termed " toxine." 

(2) Estimation of the power of the toxine by inoculation of guinea- 
pigs to determine the minimal dose which will produce death. 

Behring denotes as a normal poison a toxine solution of which 
0-01 cc. is sufficient to kill a guinea-pig weighing 250 gm. in four 
days. Of this normal toxine (d.t.n.) 1 cc. will kill one hundred 
guinea-pigs. This is the toxine unit and has a working value of 
25,000/ a toxine ten times as strong is expressed thus d.t.n. 10 : 
one ten times weaker P - T f- 

(3) Production of an antitoxic serum in a susceptible animal 
(horse) by repeated toxine injections as described above (p. 72). 

(4) Estimation of the antitoxic power of the serum by determin- 
ing how much is required to protect against the normal diphtheria 
toxine unit (d.t.n.). If, for instance, the serum is found to contain 
enough antitoxine in 1 cc. to prevent a fatal result with the injec- 
tion of 1 cc. of the toxine (d.t.n.), it is called a normal diphtheria 
antitoxine (d.a.n.). The amount of antitoxine that is required to 
protect 25,000 gm. weight of guinea pigs from the minimal fatal dose 
of the poison (d.t.n.), is termed an immunity unit (i.e.). 

To cure a person ill with diphtheria 600 to 1,800 i.e. are required 
contained in 2 — 6 cc. of serum ; therefore the strength of the serum 
used is d.a.n. 300. 

Ehrlich has propounded the idea that the antitoxines are exactly 
identical with the portions of the proteid molecule to which the 
toxines become bound in the production of disease ; the poison- 
susceptible parts of the molecule (toxophoric) are termed "side 

1 That is, 25,000 grammes weight of living guinea-pig. 


chains" (seitenketten). When a toxine is introduced in small 
quantities, some of the " side chains " unite with the toxine, 
are thrown off from the cell and at once replaced. This repair 
takes place more and more rapidly as the side chains are more 
frequently torn off by the toxine. Finally the side chains are 
produced in greater quantity than there is toxine to fix and they 
then appear in the serum as antitoxine. 

The production of antitoxine is apparently associated with an 
increase in the white blood corpuscles. 

For a summary of the present knowledge of antitoxine and 
the theories of immunity the reader is referred to the article by 
Prof. Bitchie, Journal of Hygiene, April, 1902, and to the summary 
by Armit in the British Medical Journal, April 2, 1902. 

Antibacterial Sera. — In certain diseases the production of 
immunity is associated with the production of bodies in the serum 
which possess a destructive action upon the bacteria themselves. 
These bodies may be shown to be present in various ways : — 

(1) Agglutination (Gruber and Durham, Widal). 

(2) Bacteriolysis : Pfeiffer's reaction. 

(3) Inhibition of growth (Wright). 

Fig. 31. — Widal Blood Pipette for collecting Blood for Agglutina- 
tion Reaction. 

(1) Agglutination. — Two or three drops of blood of the immunised 
animal are collected in a tube having a bulb in the centre and 
capillary ends (fig. 31) ; the blood is allowed to clot and the serum 
collected in the capillary end opposite to that by which the blood has 
been introduced. A cultivation of the organism in broth is taken and 
a drop of the diluted serum added, the reaction being carried out in 
a small test tube. If the organism be, say, B. pyocyaneus and the 
animal was immunised to B. pyocyaneus, a well marked precipitate 
soon occurs. If instead of the test tube the reaction is carried out 
on a hanging drop slide and watched under the microscope the 
motile organisms will be seen to lose their motility and become 
aggregated into clumps. This reaction is used in the diagnosis of 
typhoid fever. The serum obtained from the suspected case is tested 
in various dilutions— 50 per cent., 5 per cent., and 0-5 per cent. — 
and the effect noted in half an hour. The dilutions are made by 


diluting the serum with sterile broth by means of a capillary pipette, 
and then mixing equal loopfuls of typhoid culture and serum. 

Two pipettes are required : a 90 cm m. and a 10 cram. The 
dilutions are made as follows : — 

(1) 50 per cent. : one loopful of the broth culture is mixed with 
one loopful of the serum to be tested on the coverslip. 

(2) 5 per cent. : 90 cram, of sterile broth are mixed with 10 
cmm. of the serum, and a loopful of the mixture of serum and broth 
added to a loopful of the broth culture on the coverslip. 

(3) 0-5 per cent. : 90 cmm. of broth are added to 10 cmm. of 
the 10 per cent, solution ; and equal loopfuls of this and the 
broth culture mixed on the coverslip. 

By the above method the manipulation of the cultivation is 
limited to the platinum loop. 

The culture used should be a twenty-four-hours-old broth culti- 
vation, and a time limit of half an hour observed in estimating the 

(2) Lysogenic Action. Pfeiffer's Reaction. — Bacteriolysis. 

The culture of the organism to be tested is mixed with the 
immune serum, and the mixture injected into the peritoneal cavity 
of a healthy animal. Small quantities of fluid are removed from 
time to time for observation ; the organisms are observed to become 
swollen, contorted and finally dissolved. 

If an animal A be immunized to another's (B) red blood corpus- 
cles by the injection of increasing doses of washed red discs, the 
serum of the injected animal (A) will eventually be found to cause 
laking (haemolysis) of the red corpuscles of B when serum A is 
added to the blood of B in a test tube at body temperature. 

(3) Inhibition of Growth. Wright's Method. — The cultivation of 
the organism to be tested is mixed with various dilutions of the 
serum and melted gelatin. The mixture is introduced into capillary 
tubes and the presence or absence of colonies noted after incubation. 
The immune serum prevents the development of the corresponding 
organism. Control experiments without serum addition should 
always be made. 

We are now in a position to review the various theories of 
immunity that have been advanced from time to time to explain 
the foregoing phenomena. 

Pasteur suggested the theory of exhaustion, by which he 
endeavoured to explain the production of immunity as due to the 


using up by the infecting organisms of some portion of the tissues 
especially adapted to their growth ; and that when this hypothetical 
proteid fragment was exhausted the organisms could no longer live 
or find substances fitted for their activity, and therefore gradually 

This supposition is entirely negatived by the production of passive 
immunity by the injection of antitoxic sera, which can hardly exhaust 
the tissues of the injected animal. 

The Theory of Retention supposes that products inimical to the 
bacteria present are retained in the animal body, and that due to 
their presence the organisms die out, just as they do in an old 
cultivation in a test tube. Such a suggestion is, however, at variance 
with the main facts given above, and. is also opposed to the fact that 
death of an animal can take place by an overdose of toxine, although 
its serum is antitoxic for other animals. 

Phagocytosis. — Metchnikoff, who first advanced this theory, 
which is particularly related to the question of natural immunity, 
supposes that certain cells of the blood, termed phagocytes, espe- 
cially function as destroyers of bacteria. Of these cells two main 
varieties are present in the human subject : (a) polymorphonuclear 
leucocytes or microphages, and (b) the larger varieties of connective 
tissue cells endowed with amoeboid movement (macrophages). 
These cells are endowed with the power of ingesting foreign bodies, 
retaining them in their protoplasm until they are either wholly 
or partially digested, when if the phagocyte retains its vitality 
the remains are extruded. Such a phenomenon can be directly 
observed by keeping amoeboid cells of this class upon a warm slide 
in the presence of bacteria. The bacteria may be watched during 
the whole process of engulfing, &c, and are to be seen within the 
plasm of the cells. The phagocytes will be seen to move in the 
direction of the organisms, impelled probably by the secretions of 
the bacteria; at other times the cells recede as if to withdraw 
beyond the influence of a too powerful poison ; these movements 
are severally known as positive and negative chemiotaxis. 

Metchnikoff observed that in a susceptible animal these phago- 
cytic movements were slower, more ill-defined, or were absent, 
while in an animal possessed of a high degree of immunity the 
cells were relatively more active and ingested the bacteria with 
apparent ease and avidity ; moreover, he also observed that the 
cells of a susceptible animal acquired greater power of dealing with 


bacteria by this process of phagocytosis in proportion to the degree 
of immunity conferred by artificial means, in fact as immunity 
increased so did the phagocytic power of the cells. Natural 
immunity, therefore, would be due under this conception to a 
relatively robust and active state of the phagocytes, while suscep- 
tibility would result from a slow and indifferent phagocytic power. 
The phagocytes have, as it were, become more and more educated 
in the hunting of bacteria. 

It is, however, difficult to entirely reconcile the facts given above 
with the production of immunity by the injection of toxines without 
the bacterial bodies themselves. That a large number of bacteria 
are destroyed by the phagocytes is undoubted, and, moreover, in 
certain diseases in which bacteria are present in the blood a marked 
phagocytosis is developed, but the presence of such an extra 
development of white blood cells appears to be more in the nature 
of a concomitant phenomenon than the means by which immunity 
is effected. 

Natural Immunity. — We have so far considered the question 
of immunity from the standpoint of the pathological phenomena 
concerned in the resistance and susceptibility to various disease- 
producing bacteria. The number of pathogenic bacteria is, however, 
small when compared with the numerous species which exist as 
saprophytes, and which do not, even when introduced into animals, 
produce symptoms of poisoning. 

But animals are not always susceptible to even pathogenic 
organisms, for instance, the common fowl is highly resistant to 
tetanus, the common mouse is immune to tubercle, while the field 
mouse is susceptible, the guinea-pig is resistant to the pneumococcus, 
while the rabbit is peculiarly susceptible, and so on. 

Tn some cases it has been shown that the animal naturally 
immune has some antitoxic power in its serum, and that the blood 
is also antibacterial, but this is only in a few cases and is not 
sufficiently generalised to account for the immunity possessed by 
many animals. 

Animals, therefore, possess a natural immunity to certain 
diseases which may be due (a) to the power of animal tissues 
generally to destroy bacteria (b) to the ease with which the 
toxines of the infecting organisms are neutralised in the body. 
Both of these processes may be in operation at the same time and 
by appropriate means one or other may be so lowered by artificial 


means that the animal easily develops the disease. Thus Pasteur 
found that the fowl, normally immune to anthrax, became suscep- 
tible when its body temperature was reduced by cold water. Guinea- 
pigs and rats kept in an atmosphere of sewer air showed lowered 
resistance towards diphtheria bacilli. Animals, such as rats, &c, 
which have been fatigued by exercise in revolving cages, are found 
to be much more susceptible to staphylococcal injections than 
control animals. 

Immunity is therefore in some way concerned with the normal 
functions of the body, but at present very little is known of the 
subject. It is probable that fresh light will be thrown on the 
subject by the large amount of enquiry that is at present going 
forward relative to the anti-bodies produced during artificial im- 
munization, and it is also possible that the property of natural 
immunity to many diseases is the expression of a gradually 
developed tolerance to the attacks of micro-organisms evolved 
over long periods of time, and produced in a manner analogous 
to artificial immunization. Immunity is, moreover, transmitted 
from mother to offspring. 


Pathogenic Bacteria of the Mouth. 

From time to time various pathogenic bacteria are to be found 
inhabiting the mouth, and may be obtained from the fluids of that 
cavity, sometimes by means of cultures alone, but at any rate 
with certain species, best by the inoculation of animals with saliva. 

Most of the bacteria thus obtained prove to be members of well 
known species which are generally associated with disease and 
pathological conditions in other regions of the body, a few are 
members of species as yet little studied, whilst a good many 
described by various authors have occurred in isolated cases only. 
It is impossible, in many cases, to find an adequate description of 
many of this latter series, and the task of connoting all the 
evidence is particularly difficult ; some of the organisms may well 
belong to known species, modified perhaps by their residence in the 
mouth in such a manner as to render them difficult to identify in 
the first cultures obtained. 

In noting some of these bacteria in the present chapter, the 
description given by the observers who originally described them 
have been rigidly adhered to, and the whole of the data obtainable 
given. This has been done with two reasons : firstly, to make the 
chapter as complete as possible ; and secondly, by collecting the 
various scattered researches to enable other workers to confirm or 
disprove the various statements made. Special note is made of 
any of these organisms I have met with myself during a somewhat 
extended series of researches in the last seven years. Many of the 
pathogenic bacteria which occur in the mouth are apparently living 
in a state of ceco-parasitism, ready under favourable circumstances 
and environment to act as the liberating impulses of disease. 

Amongst the most commonly present the pneumococcus is 
perhaps the most frequent ; its pathogenicity varies within wide 
limits. It is somewhat interesting to note that this organism, dis- 
covered independently by Frankel, 1 Pasteur' 2 and Sternberg, 3 was first 

Zeit. fiir Klin. Med., 18S5. 

Compend. rend Acad, des Soc, Paris, xcii., p. 159-165. 

National Board of Health Bull., vol. ii., 1882. 


isolated and described by inoculating rabbits with saliva. Pasteur 
was searching for the organism associated with rabies, Sternberg 
for the cause of yellow fever. 

Another pathogenic organism whose presence in the mouth is 
more general than is often appreciated by dental surgeons, and 
which appears in about 33 per cent, of all persons exposed to infec- 
tion, is the Klebs-Lceffler or diphtheria bacillus, and moreover the 
spread of this disease is largely due to the transference of the 
organism from one individual to another, more particularly children. 
So much is this the case that notwithstanding the increase of 
sanitary knowledge and the application of general principles of 
hygiene to everyday life, the disease, once more common in rural 
than urban districts, has now become a disease more associated with 
town than country areas, and shows a most striking relationship to 
the progressive aggregation of children for educational purposes. It 
is therefore a disease which should be borne in mind by all dental 
surgeons. In all mouths, and with no exception as far as I can 
ascertain, a streptococcus is a normal inhabitant, but apparently 
exists as a distinct species of a non-virulent type, although at times 
true pathogenic streptococci are to be met with. B. coli communis 
is also to be found rarely, while the tubercle bacillus may be found 
in the subjects of tuberculosis of respiratory tract. The bacillus 
of blue pus, B. pyocyaneus, is found in a limited number of cases, 
as is the Micrococcus tetragenous and rarely the Streptothrix actino- 
myces. These more important pathogenic organisms will be described 
first and following them a second group, comprising pathogenic 
organisms observed and described by various authors as peculiar to 
the mouth. 


This streptococcus is the organism associated with erysipelas 
and general pyaemia. It has been found also in a large number of 
other pathological conditions, such, for instance, as infective endocar- 
ditis, puerperal septicaemia, acute infective periostitis, &c. Associated 
with other pathogenic organisms it is sometimes found in diphtheria, 
bronchitis, pneumonia (especially the type known as " septic 
pneumonia"), as a secondary infection in phthisis, &c. In many 
cases of obscure febrile type the organisms are to be obtained from 
the circulating blood. 



Fig. 32. — Streptococcus pyogenes in .Blood, x 1,000. 

Fig. 33. — Streptococcus pyogenes. 
Twenty-four hours' agar cultivation. Stained Gram. x 1,000. (From 
Washbourn and Goodall's "Infectious Diseases.") 



The streptococci are among the most widely distributed of the 
pathogenic cocci, and are as a general rule present in all the more 
acute suppurative conditions found in man. 

Varieties. — Formerly the streptococcus of phlegmonous erysi- 
pelas, and the S. pyogenes were considered different species, but 
from a series of extended researches it is now generally admitted 
that the two species are identical. 

Fig. 34.— Steeptococcus pyogenes. 
Twenty-four hours' agar cultivation. -§. 

Petruschky placed this beyond doubt, by obtaining a virulent 
streptococcus from a purulent peritonitis in a patient never having 
suffered from erysipelas. He then inoculated the cultures into two 
individuals suffering from cancer, who had never had erysipelas, and 
produced a definite attack of erysipelas. 

Linglesheim, 1 by a long series of carefully-conducted researches, 

1 Zeitscli. fur Hygiene, Bd. x., p. 331. 


came to the conclusion that the streptococcus normally resident in 
the mouth was of a distinct species to the organism causing disease. 

Other observers, however, have held that all streptococci, 
wherever obtained, are simply different races of the pathogenic 
streptococcus, and may be [raised to the same standard of virulence 
by appropriate means. This question is considered more at length 
below, and I will only remark in passing that it cannot be definitely 
stated that all the streptococci found occurring in disease are of the 
same species. 

Morphology. — Cocci, 0*4 — 1 n in diameter, generally united in 
chains of four to twenty or more individuals, the longer chains are 
generally formed in liquid media. When rapid division is taking 
place the appearance of a chain of diplococci is produced. According 
to Sternberg, the cocci may occasionally occur as diplococci in culture 
media. Sometimes the cocci become so much elongated that the 
appearance of bacilli is produced. 

In old cultures particularly, some of the elements of the chains 
appear enlarged and swollen and greatly exceed the size of the rest 
of the cocci in the chain ; the bodies have been termed arthrospores 
by Hueppe and Du Bary, and are thought to be more resistant than 
the purely vegetative forms. Other bacteriologists consider them to 
be involution forms. No true endospores are formed, the cocci are 
not motile and do not possess flagella. 

Staining Reactions. — Stains well with the ordinary aniline dyes 
and by Gram's method. Old cultures, and especially cultures on 
potato, show irregular staining. 

Biological Characters. — An aerobic, facultative anaerobic, non- 
motile pathogenic streptococcus. Spore formation not known, not 
motile, no flagella present, does not liquefy gelatin. Optimum 
temperature 37*5° C, but will grow at 16 J to 18° C. Thermal death 
point 54° C. for ten minutes. 

Gelatin Stab, 22° C. — Growth slow ; in three days a line of small 
spherical, translucent, whitish colonies, giving a beaded appear- 
ance. Surface growth weak, flat, and edge entire. No liquefaction 

Gelatin Streak, 22 C. — A series of small, flat, whitish colonies, 
rarely forming a continuous streak. 

Gelatin Shake, 22° C. — A cloud of minute spherical colonies are 
seen distributed throughout the medium in three to four days. No 
gas is formed. 


Agar, 37*5° C. — A series of grey translucent flat colonies develop 
along the- streak, in places becoming confluent. The edges of the 
colonies when observed with a lens are seen to be composed of 
loops of chains of cocci. 

Blood Serum, 37*5° C. — Minute grey-white colonies similar to agar. 

Potato. — Little growth occurs, and in forty-eight hours at 37*5° 
most of the cocci have become swollen and involuted. 

Broth. — Development somewhat slow at 37'5° C. The fluid 
remains clear, and numerous thin flat flocculi form, which tend to 
settle to the bottom or to the inclined side of the tube ; when blood 
serum or ascitic fluid is added to the broth, development is more 
rapid. A faint acid reaction does not prevent the growth, although 
the organisms grow best in an alkaline or neutral medium. No 
indol formed. 

Glucose Broth. — Acid, no gas. 

Litmus Milk, 37*5° C. — Well marked acid reaction in two days, 
later the milk may be coagulated, but this is by no means constant. 

The vitality of the cultures is not great on liquid media. They 
are best kept on solid media, and withstand drying for some time. 

Pathogenesis. — The streptococcus varies considerably in the 
amount of pathogenic power possessed by various races, and is not, 
as generally obtained, very virulent for animals. Mice and rabbits, 
if inoculated with virulent culture, die in twenty-four hours to six 
days, of general septicaemia, the organisms are present in large 
numbers in the heart blood and in the various organs. If the culture 
be less pathogenic, disseminated or metastatic abscesses are found 
distributed about the various organs. 

Marmorek 1 has shown that the initial virulence possessed by 
streptococci obtained from various pathological conditions arising in 
the human subject, may be raised to a great degree of virulence by 
the rapid passage through rabbits, one animal being inoculated with 
the blood of another just dead of streptococcal infection. The cul- 
tures were made upon a special medium, consisting of three parts of 
human blood serum to one of broth. 

Bulloch 2 also produced enormous exaltation of virulence by 
passage through rabbits. The culture on ascitic fluid- broth, which 
originally had a m.l.d. 1 of Ol ccm., was increased in virulence to 

' Ann. de VInst. Pasteur, t. ix., No. 7, 593. 
- Lancet, April 11, 1896. 


such an extent that 0-000001 ccm. was the m.l.d. Many other 
workers have confirmed these results. Widal and Besancon 
found that a streptococcus which originally possessed no virulence 
became pathogenic when inoculated with Bacillus coli communis, 
and that subsequent cultures could be raised in virulence by 
Alarmorek's methods. These observers also found that the strepto- 
cocci obtained from the mouth of a small-pox patient were non- 
virulent, whereas those present in the blood exhibited a considerable 
degree of pathogenic power. 

Fig. 35. — Streptococcus pyogenes. 
From twenty-four hours' broth cultivation. Stained Gram. x 1000. 


(Streptococcus brevis.) 

Any one who has made cultivations from the mouth cannot but 

have been struck with the frequency of streptococci in the cultures. 

In all mouths, healthy or unhealthy, clean or dirty, I have never 

failed to obtain the streptococcus. Not only is it present in the 

mouth proper, but it exists in the antrum of Highmore, in the 

Eustachian tube, nose, and middle ear. In these situations it 

occurs typically as diplococci massed around the dead squamous 

1 Minimal lethal dose. 


Fig. 36.— Streptococcus brevis of Mouth, massed round Epithelial 

Cell, showing Diplococcal Form. 

Stained Gram, x 1,000. (Washbourn and Goadby, Trans. Odont. Soc, 1896.) 

Fig. 37.— Streptococcus brevis. 
Agar cultivation at twenty-four hours. Stained Gram, x 1,000 


epithelial cells (fig. 36). It is by no means confined to the human 
mouth, and I have observed it in the mouths of monkeys, dogs, 
rabbits, and guinea-pigs. 

The diplococci form of this streptococcus can be easily seen in 
almost any coverslip preparation made from the mouth direct. A 
cover glass specimen is made by smearing saliva, scraped from the 
buccal sulcus with a platinum loop, on to the cover glass. The 
film is allowed to dry and then stained with carbolic-methylene 
blue or other stain. The diplococci will readily be recognised 
massed around and adhering to the squamous epithelial cells, some 
of the diplococci having the appearance of short bacilli owing to the 
elongated and somewhat pear-shaped form of the cocci. The 
question of the identity of these diplococci with the streptococcus 
may be proved in the following way : clean coverslips are smeared 
with melted agar to which a little saliva containing some epithelial 
cells has been added ; when dry, the small coverslip-plate is 
cemented to a hanging drop slide (a slide with a glass ring cemented 
to it, see fig. 13) and fixed in place with a little Canada balsam. The 
preparation is then placed on the stage of the microscope and an 
epithelial cell sought for with the diplococci attached. The micro- 
scope with the preparation in situ is then placed in the incubator at 
37*5 3 C. for twenty-four hours. At the end of this time the pre- 
paration is examined, when the diplococci will be found to have 
developed into colonies which surround the cell and in which the 
streptococcal chains may be easily seen. With a little care one 
colony can be selected and marked, and cultures on broth and agar 
made from it as well as coverslip preparations. The culture tubes 
will show a good growth of streptococci. 

This method of obtaining a pure culture of the mouth strepto- 
coccus is somewhat tedious, and the method adopted by Dr. 
Washbourn and myself is much less difficult. Broth cultures are 
made by adding a loopful of saliva to a tube of nutrient broth ; the 
tube is then incubated for twenty-four hours, at the end of which 
time the tube will contain an impure culture of streptococci and 
other organisms. An agar tube is now inoculated from the broth 
tube and the agar tube incubated at 37° C. In eighteen to twenty- 
four hours the sloped surface of the agar tube will be found to be 
covered with a number of small grey-white colonies, which, trans- 
ferred to another tube, will give a pure culture of the mouth strepto- 
coccus. A little care must be exercised in picking out the colonies, 


as other organisms are often also present, but there is generally no 
difficulty in obtaining a pure culture. The explanation of the ease 
with which a culture can be obtained is that the mouth strepto- 
coccus grows with great rapidity in almost all media, to the 
exclusion of other organisms. 

The streptococcus obtained from various mouths often differ 
slightly in their cultural characters, but in their general behaviour 
they conform to the type of von Lingelsheim's 1 Streptococcus 
brevis, as noted by Dr. Washbourn and myself. 2 Since that 
paper was published I have obtained the streptococci from 150 
consecutive mouths examined ; all the cultures obtained conformed 
to the general characters given below. All of them produced an 
acid reaction when grown in carbohydrate media (broth to which 2 
per cent, of lactose, maltose, glucose, dextrin, cane sugar, or starch 
had been added). The starch and cane sugar media require the 
longest to develop the acid reaction, whilst in the other solutions 
the reaction is often strongly acid in six hours. 

The streptococci also clot milk into a solid mass in forty-eight 

In the paper quoted above it was noted in connection with the 
mouth streptococcus that among other characters the few inocula- 
tion experiments performed confirmed von Lingelsheim's view that 
the mouth streptococcus differed from the Streptococcus longus in 
not being pathogenic for guinea-pigs, rabbits and mice. Other 
observers are inclined to the view that the streptoccocus of the 
normal mouth is the ordinary pathogenic streptococcus which occurs 
in pyaemia and phlegmonous erysipelas. Pathogenic streptococci 
undoubtedly do occasionally occur in the normal mouth, as various 
observers have shown — a fact that considerably complicates the 
problem. The pneumococcus occurs in normal human saliva 
in a distinctly pathogenic condition, so much so that a rabbit, 
an animal particularly susceptible to the pneumococcus, often 
dies subsequent to an inoculation with saliva, the pneumo- 
coccus being found in the blood after death. One of the races 
of pneumococci obtained from the saliva by Washbourn 3 and Eyre 
were, however, apparently living in a saprophytic condition, and 

i Zeitschrift fiir Hygiene, Bd. x., p. 331. 

2 Trans. Odont. Soc, June, 1896. 

3 Brit. Med. Jour., Nov. 4, 1899. 


their virulence was of low value until the organisms had been pssaed 
through the bodies of many animals ; even then the pathogeuicity 
of the species soon ran down when grown upon artificial media, 
whilst other races of pneumococci obtained from the rusty sputum 
of pneumonia retained their virulence for a considerable time. 
Virulent diphtheria bacilli may be present in normal mouths as 
well as other pathogenic organisms, and we may certainly also 
conclude that the streptococci of a pathogenic nature met with 
in the mouth from time to time are stray individuals of another 
species accidentally present, and not the common mouth inhabitant. 
This question of the identity of two presumably different organisms 
is a much wider question than the particular case of the mouth 
streptococcus ; thus, for instance, B. coli communis and B. typhi 
abdominalis, B. diphtherias and the B. of Hoffmann, B. subtilis 
and B. anthracis, to mention only a few examples, are each 
related to the other in their cultural peculiarities, method of stain- 
ing, &c, and somewhat minute differences are relied upon to 
differentiate the organism from its simulator. It is of course 
possible that the streptococcus of the mouth is a degenerate non- 
pathogenic and saprophytic variety of the Streptococcus longus, 
and that under some favourable conditions it may invade the tissues, 
as in severe scarlatinal angina, and produce serious results. On 
the other hand the mouth streptococcus may be a different species, 
having certain characters in common with the Streptococcus longus 
it is true, but differing from it in others, among which is its viru- 
lence. Lmgelsheim 1 was the first to point out that the streptococcus 
obtained from the normal mouth differed from the Streptococcus 
longus. Thus it was not pathogenic for rabbits or mice, it ren- 
dered broth uniformly turbid, and the chains on this medium were 
shorter than those of the Streptococcus longus and it caused a slight 
liquefaction of gelatin, and Lingelsheim therefore considered it a 
distinct species and named it the Streptococcus brevis, from the 
short chains formed on broth cultures. Marmorek,' 2 in opposition 
to this, looks upon all streptococci as simple varieties of the same 
species, which can all be raised to a uniform type by appropriate 
means, although various strains of streptococci obtained from 
different pathological conditions of the human subject behave 
differently when injected into animals. 

1 Loc. cit. - Loc. cit. 


In the paper already quoted 1 the following conclusions are 
given : — " The streptococcus occurring in the normal mouth agrees 
with the S. brevis of Lingelsheim, and can be distinguished from the 
streptococcus of disease by its biological and morphological charac- 
ters. It must be looked upon as a distinct species for the present, 
although ultimately this view may prove to be incorrect, for it is 
possible that further researches may enable us to convert the 
Streptococcus brevis into the Streptococcus longus. This, however, 
has not hitherto been accomplished. We think that the dis- 
crepancies of different observers who have investigated the question 
are partly due to the fact that the pathogenic Streptococcus longus 
is sometimes accidentally present and has been mistaken for the 
normal streptococcus of the mouth." 

Subsequent research tends to confirm these conclusions, and for 
the present the S. brevis of the mouth is to be regarded as a distinct 
species and as the most constant of all mouth organisms. In per- 
fectly clean and healthy mouths it is often the only organism 
met with. 

Various other pathogenic organisms have been stated to be 
present in the normal mouth. Biondi 2 particularly gives a list of 
five organisms of this class, which require notice, Of the five 
organisms in Biondi's list two were only met with once, in each 
case by inoculating an animal with saliva, the organism being found 
in the resulting abscess. These two organisms then (Coccus sali- 
varius septicus and Staphylococcus salivarius pyogenes) can hardly 
be called true mouth bacteria. 

The third on Biondi's list (Streptococcus septo-pyeemicus) is said 
to be indistinguishable from the streptococcus of pyaemia and 
erysipelas in its cultural peculiarities and its pathogenic action on 
animals, and there seems no reason to doubt that this streptococcus 
was the S. pyogenes which we have seen is at times present in the 

The fourth organism described by Biondi is the Micrococcus 
tetragenous, whilst B. salivarius septicus has since been shown to 
be the same as the Diplococcus pneumoniae. 

1 Loc. cit. 

- Zeitschrift fur Hygiene, Bd. ii., 1887, p. 194. 



The Staphylococcus aureus, or golden coccus, first carefully 
described by Eosenbach in 1884, is commonly present in suppura- 
tion, abscesses (acute), boils, carbuncles, osteomyelitis, ostitis, &c, 
and occasionally in puerperal fever, infective endocarditis and 
pyaemia. In cultures from septic throats it is often present and is 
commonly found as a contamination in cultivations made from the 
throats of persons ill with diphtheria. Outside the body it may be 
found in air, soil, or water, but most frequently in dust, especially 
the dust of hospital wards. 

The staphylococcus may occur in the eyes, ears, nose, mouth, 
and especially under the finger nails, whilst they are said to occur 
occasionally in the faeces of children. 

I have several times obtained the staphylococcus aureus in pure 
cultivation from acute abscesses involving the roots of " dead teeth," 
but it is by no means always present, for in forty cases of oral sup- 
puration around teeth they were only present three times, twice in 
pure culture. On two occasions I have found a pure culture of 
staphylococcus aureus in antral suppuration, but even in this region 
it is far from common. 

In neglected and dirty mouths these organisms are occasionally 
to be found, in healthy mouths they are not often found. 

The majority of observers agree that the S. aureus is by no 
means always present in the mouth, thus : Netter 1 only found S. 
pyogenes aureus in seven out of 127 individuals examined ; Vignal- 
and Miller 3 only found the pyogenic cocci occasionally ; Black, 4 on the 
other hand found S. aureus in seven out of 10 cases examined. My 
own researches tend to confirm those of Netter and Miller, the 
S. aureus occurring in about 10 per cent, of all cases examined. 
On looking through the notes of the examination of 1,000 mouths, 
I find staphylococcus albus occurs eighty times, about 10 per cent. 
In these cases there was no special search made for the organisms, 
but the yellow colonies were observed in the ordinary process of 
cultivation, isolated and grown on the various test media. 

A fact that may to some extent explain the rare occurrence of 
the staphylococcus aureus in the mouth is the bactericidal power of 

1 Revue (V Hygiene, 1889, No. 6. 

-Arch, de Physiol, normal et path., 1886, No. 8. 

3 "Micro-organisms of the Mouth," p. 265. 

4 Trans. III. St. Dent. Soc., 1886. 


the saliva experimentally proved by Sanarelli. Sanarelli 1 filtered 
saliva through a Pasteur-Chamberland filter and tested the filtrate 
in the following way : — 

A small quantity of a pure cultivation of staphylococcus aureus, 
just as much as could be taken up on the point of a platinum needle, 
was added to 10 cubic centimetres of saliva sterilised by filtration. 
After mixing, plate cultivations were made, and the Dumber of 
organisms present determined. Plate cultures were then made at 
intervals, and by this means it was found that the number of 
colonies developing gradually diminished, and in thirty-six hours no 
staphylococci were present. 

If, however, a whole loopful of the cultivation was added, the 
plate cultivations still showed a diminution in the number of organ- 
isms present during the first two days, but after this the number 
again increased, until the colonies were uncountable. 

It follows, therefore, that the saliva possesses a certain bacteri- 
cidal power which is able to deal with small quantities, or isolated 
organisms, but that this power is quantitative, and that it is insuffi- 
cient to deal with a large number. In this respect the action is 
similar to that of blood serum and other body fluids. 

It is interesting to note that pneumococci were apparently not 
affected by the action of the saliva, and grew from the first. 

Morphology. — Eound, spherical or oval cocci, 06 — 0*9 /" diameter, 
occurring in clusters or singly and in chains, showing a slight 
flattening at the point of mutual apposition. Not possessed of 
flagella. According to Du Bary and Hueppe arthro spores are 
formed ; these consist of large swollen elements 1-0 to 1*5 /* wide, 
which stain deeply, other observers are inclined to regard such 
forms as degenerate cocci, and they are known also as " involution 
forms." No endogenous spore formation is known to occur. 
Capsule not stainable. 

Staining 'Reactions. — Eetains the stain of Gram's method, and 
stains easily with the ordinary basic aniline dyes, carbol methylene 
blue, gentian violet, &c. 

Biological Characters. — Non-motile, aerobic, facultative anaero- 
bic, chromogenic coccus, with well marked fermentative and patho- 
genic powers. 

Gelatin, 22° C. — Plates in forty-eight hours white punctate 
colonies, which rapidly liquefy the medium, microscopically 
granular, dark brown, entire. 

1 Centr.filr Bakteriol., Bd. x., 1891, p. 817. 


Gelatin Stab, 22° C. — Growth to bottom of stab with rapidly 
following liquefaction to depth of puncture, cone-shaped liquefaction, 
with thick golden-yellow precipitate and thick flocculi in fluid. 

Gelatin Streak, 22° C. — Well marked groove of liquefaction in 
three days with flocculent yellow precipitate. 

Agar Streak, 37*5° C. — Moist irregularly creuate, raised, yellow. 
Pigment formed at 37-5° C, but best at 22° C. 

Agar Stab, 37'5° C. — Growth to bottom of puncture. 

Potato. — Yellow, moist and well marked development at 37*5° C. 
and 22° C, most pigment at 22° C. 

Blood Serum (coagulated), 37*5° C. — Similar to agar, but colonies 
may be smaller, no liquefaction occurs. 

Broth, 37-5° C. — General turbidity in twenty-four hours at 
37-5° C, a heavy yellow-brown precipitate gradually settles, often 
in flakes. 

Litmus Milk, 37'5 D C. — Acid reaction in twenty-four hours, 
coagulation of casein in two to four days with subsequent digestion 
of coagulum. 

Peptone Water, 37 # 5 J C. — Similar to broth. All cultures give 
off a sour acid smell. Optimum temperature, 37 5° C. 

Carbohydrate Media — Well marked acid reaction. The liquefy- 
ing enzyme may be separated from the cultures by precipitating 
with alcohol, and will produce liquefaction independently of the 
organisms. The thermal death point of this organism is between 
56° and 58° C. for ten minutes, when in the moist condition ; when 
dry, however, a temperature of 90° to 10(P C. acting for the same 
time is required. 

The resisting power to antiseptics has been tested by a large 
number of observers : Gartner, Berhing, Tarnier, and Vignal, all 
agree that the organism is destroyed by exposure to 1 : 1,000 mercuric 
chloride for one to three minutes. Abbot, 1 however, has shown that 
in the same culture all the cocci are not of the same resistance, and 
that occasionally there may be forms that will resist an exposure 
of ten, twenty, or even thirty minutes, although the majority are 
destroyed by the same solution in two minutes. 

Chromogenesis variable, some cultures giving a marked colour, 
others only a faint yellow. After some time at 37*5° C. the colour 
tends to become a dirty buff. 

1 Bull. Joints Hopkins Hosp., vol. ii., 1891. 


A variety of S. pyogenes aureus which does not liquefy gelatin 
is said to exist, the colour being darker and less brilliant. 

Pathogenesis. — Well marked virulence in some specimens, but 
variable and often lost in saprophytic condition. 

Garre caused a well marked carbuncle on his own arm by 
applying a virulent culture of S. pyogenes aureus to the uninjured 

Bumm produced abscesses by inoculating the cocci suspended 
in normal saline under his own skin. 

Becker and others found that by injecting the staphylococcus 
into the circulation of animals after fracturing or crushing a bone, 
osteomyelitis resulted. 

Eibbert and Eosenbach 1 produced ulcerative endocarditis by 
injecting the cocci into the circulation of animals whose cardiac 
valves were injured by passing a sound down the carotid artery ; 
many other observers have confirmed these experiments. 

The injection of small quantities of staphylococcus aureus under 
the skin of animals may often produce no effect ; fairly large 
quantities, however, generally produce a marked localised abscess 
which eventually clears up. The injection of small quantities into 
the blood stream (intravenous injection) generally produces death 
in rabbits and guinea-pigs, the most characteristic lesions occurring 
in the cortex of the kidney, where rows of small abscesses, observ- 
able by the naked eye, occur. These abscesses may clear up 
without death of the animal, and Morse 2 has shown that in such 
cases, besides the plugging of the small vessels with masses of cocci, 
an interstitial growth of connective tissue has occurred (interstitial 
nephritis) which may be reproduced by injection of the toxines of 
the organism. It certainly appears from Morse's paper that some 
amount of toxine development by the staphylococci occurs in the 
tissues, leading to development of fibrous tissue. 

In septic wounds in man it is often possible to obtain a culture 
of the staphylococci from the blood, although a general pyaemia 
rarely results from infection with this organism, but kidney infection 
and metastatic abscesses of joints may occur. There is generally 
little difficulty in obtaining cultural evidence of the presence of the 
staphylococcus aureus in pus or other suspected fluid. A small 

1 Fortsch. der Med., 1886, p. 1. 

- Joum. Experimental Medicine, vol. i. , 4, p. 618. 



amount of the pus should be collected in a sterile pipette (see fig. 38) 
and a drop or two smeared over the surface of an agar tube ; in 
twenty-four hours at 37° C. well marked development occurs, and the 
yellow colonies may be easily recognised, isolated and tested on the 
usual media. Blood is treated in a similar manner, but should be 
well diluted with sterile broth. 

Fig. 3S. — Pipette for Collecting Pus. 
The top is closed with a cotton wool plug, and the lower end broken at 
time of use and sealed up in the flame when the tube is filled. 

Several other varieties of staphylococci exist, and of these S. 
pyogenes albus is perhaps the most common, and certainly is the 
most frequently present in the mouth. In the majority of cases 
of suppuration in and about the mouth this organism may be met 

In its morphological, biological and general characters the staphy- 
lococcus albus closely follows what has been said of the S. aureus 
with the exception of the production of yellow pigment. Watson 
Cheyne is inclined to think that this staphylococcus is the most 


virulent of the two, but the general opinion is rather of the opposite 

The S. albus as obtained from the mouth generally exhibit more 
of the special peculiarities attributed to the S. epidermis albus of 
Welche in its rate of liquefaction, greater tendency to the diplo- 
coccal form, and low pathogenicity. 

The Staphylococcus pyogenes citreus, another variety of the 
staphylococcal group, differs from the other two described in the 
colour of its pigment, which is of a marked lemon-yellow ; it 
is often present in the mouth, but it is not so virulent as the 
S. aureus. 

Some authors, among them Lehmann and Neumann, are 
inclined to regard all these pathogenic staphylococci as the same 
species, and group them accordingly as Micrococcus pyogenes, 
remarking that the staphylococcal form is not sufficiently constant 
to form a class term for the species. 


Sternberg in 1880, and Pasteur later in the same year, described 
a diplococcus of pathogenic power which they had obtained from 
the saliva of normal individuals. 

In the blood of animals injected with saliva a diplococcus was 
observed, exhibiting amongst other peculiarities a well-defined 
capsule capable of being stained by appropriate methods. The 
organisms were extremely difficult to obtain in pure culture ; the 
use of "blood agar " introduced by Washbourn, however, obviated 
the difficulty, and the organism may be easily grown on this 
medium. It is to Frankel 1 and to Weichselbaum 2 that we are 
indebted for the discovery of the relationship the organism bears 
to pneumonia. The pneumococcus or micrococcus pneumonias 
crouposae, so called from its relation to that special form of 
pneumonia, is by no means confined to the disease of the lung 
but has been found in many other situations. As it enters the 
blood stream and may therefore be carried to any part of the body 
this is by no means remarkable. The pleural sacs are often 
directly infected ; a pure culture of the pneumococcus often results 

1 Zeitsch. flir Idin. Med., Bd. xi., 1886, Heft 5 and 6. 
- Wiener mad. Jahrbucher, 1886, p. 483. 


from cultivations made from pleuritic effusions. The organism may 
occur also in the meninges of the brain, setting up pneumococcic 
meningitis primarily or as a complication of pneumonia ; it has been 
found occasionally in abscesses of the vermiform appendix. The 
organism has also been observed in suppuration occurring in the 
subcutaneous tissues, joints, liver, spleen, kidneys, and in otitis 
media, infective endocarditis, pericarditis, &c. 

It occurs with amazing frequency in the saliva of normal 
individuals. Netter : found it present in 15 per cent, of healthy 
individuals. Wolfe,' 2 Claxton, 3 Friinkel, 4 Washbourn and Eyre 5 and 
many others have also noted its presence in the mouths of perfectly 
normal individuals. The organism is difficult to cultivate directly 
from the saliva owing to the presence of other bacteria ; the 
method generally adopted is to inoculate a susceptible animal, 
such as the rabbit, with saliva. If pneumococci are present the 
animal dies, and the organisms may be obtained from the heart, 
blood, and spleen. 

The cocci thus obtained differ considerably in their virulence ; 
sometimes the amount of pathogenic power is extremely small, as 
was the case with the organisms mentioned by Washbourn and 
Eyre, which required passage through the bodies of fifty-three 
animals before they attained the same pitch of virulence that another 
pneumococcus obtained from a case of pneumonia attained after 
only eight passages. On the other hand, an organism obtained 
from the mouth is often found to possess a high degree of virulence, 
and Dr. Eyre informs me that for more than two years fully virulent 
pneumococci have been present in his own mouth. The organism 
occurs typically in the rusty or prune-juice sputum of pneumonia 
and at the edge of the consolidated lung tissue. 

Morphology. — Cocci, oval or spherical in shape, generally united 
in pairs, but also occurring in chains of three or four elements, 
especially upon agar and liquid media. In the blood of inoculated 
animals, and in the sputum of acute pneumonia, the cocci are 
arranged in pairs of oval shape, and surrounded with a gelatinous 
capsule. The shape of the cocci is frequently lanceolate. 

The capsule is said to consist of a substance resembling mucin, 
and is soluble in water, and does not appear when the organism 
is grown in artificial media. 

l Loc. tit. - Weiner meet. Blatter, 1882. 

3 Med. Times, Philadelphia, 1882. * Loc. cit. 5 Loc. cit. 


Staining Reaction. — Stains well by Gram's method and by the 
ordinary aniline dyes. The capsule may be stained, in fresh 
specimens from the blood of inoculated animals, or from sputum by 
means of MacConkey's capsule stain, or by the glacial acetic acid 
method. Glacial acetic acid is poured on the film prepared in the 
ordinary way and immediately poured off, and the slip plunged 
without washing into aniline-gentian-violet. 

Biological Characters. — Aerobic facultative anaerobic, non- 
liquefying, non-motile pathogenic coccus. 

Gelatine Plates, 22° C. — Bound, grey, 1-2 mm. in four days, deep 
colonies round, entire, slightly granular microscopically. 

Gelatine Stab, 22° C. — Thread-like (filiform), later beaded, no 
liquefaction, little surface growth. 

Agar Plates, 37 '5° C. — Macroscopically like gelatin. Micro- 
scopically, deep, round, lenticular, greyish-black, coarsely punctate ; 
surface round, entire, translucent, finely punctate. 

Blood Agar Streak, 37*5° C. — Greyish, punctate, edge entire 
or slightly dentate. The colonies generally remain separate. 

Blood Serum, 37*5° C. — Slimy, transparent, no liquefaction. 

Potato, 37*5° and 22° C. — No growth, except occasionally in 
old laboratory cultures. 

Litmus Milk, 37*5° C. — Sometimes an acid reaction, with or 
without coagulation, but variable. 

Broth, 37*5° G. — Faintly turbid with a slight flocculent precipitate. 

Note. — The growth on gelatin plates and stab given above is taken from 
Lehmann and Neumann's "Bacteriology." On the other hand, Dr. Eyre 
assures me that only the non-virulent, or slightly pathogenic forms with which 
he has worked grow at all on gelatin, and even then only upon gelatin streaks, 
never upon plates or stab cultures. Fully virulent forms do not grow at all 
upon gelatin at 22° C. 

Pathogenesis. — A small quantity of an agar culture inoculated 
into a mouse, rabbit, or guinea-pig produces death in one or two 
days. Injection of saliva containing the pneumococci, rusty sputum 
from lobar pneumonia, or a piece of lung tissue from croupous 
pneumonia produces the same result. At the autopsy signs of 
general infection are present. At the site of inoculation there is 
generally a well-marked fibrinous or gelatinous exudate, often half 
an inch in thickness. The pleural, pericardial, and peritoneal 
cavities are generally full of fluid. The spleen, liver, lungs and 
other organs contain the cocci in large numbers. They are also 


present in the heart blood and general circulation — in other words, 
a general septicaemia occurs. The lungs show no distinct pneumonic 
changes, although Marli has claimed to produce pneumonia in 
animals by the injection of pathogenic cultures of the pneumo- 
coccus into the trachea. 

The cocci exhibit the typical capsulated form when stained from 
the heart blood of an animal dead of pneumococcic infection. 

The amount of immunity produced by infection with the pneumo- 
coccus is very brief, and although many observers have attempted 
the isolation of a definite toxine from the cultures of the organism, 
no success has so far rewarded their efforts ; it is probable, how- 
ever, that such a toxine does exist in rather minute quantities. 

G. and F. Klemperer showed that the serum of immunised 
animals protected animals against infection with the pneumococcus. 

Washbourn has also prepared an anti-pneumococcic serum which 
will protect against one hundred times the fatal dose of pneumococci. 

The pneumococcus is capable of attaining an enormously high 
degree of virulence, and in the experiments conducted by Wash- 
bourn and Eyre this " standard virulence " was 0*000001 of a 
loop holding 0-5 milligram of culture, this amount invariably pro- 
ducing death in rabbits, and an anti-pneumococcic serum was 
obtained of such strength that it protected against 1,000 times 
this fatal dose. Considerable benefit has resulted from the use of 
this serum in certain cases of pneumonia. Washbourn and Eyre 
are continuing their experiments with the pneumococcus in several 
directions, and have recently published further notes upon the 
Pathology of Pneumococcic Infection. 


This coccus was originally observed and studied by Koch and 
Gaffky, who found it in the lung cavities of tubercular persons. 

It is often present in normal saliva, and is not infrequently 
present in dento-alveolar abscesses. Biondi found it three times in 
fifty cases examined, Miller also has met with it frequently, as have 
Park, Vangel and Steinhaus. 

Morphology. — Cocci spherical and arranged in groups of four, 
due to division in two planes at right angles to each other. The 
individual cocci are about 1 /* in diameter ; the whole group is 
generally surrounded with a gelatinous capsule. The typical 


arrangement and the capsule are only constantly present in the 
tissues of inoculated animals. 

Staining Reactions. — Stains by the ordinary aniline dyes and by 
Gram's method. 

Biological Characters. — Aerobic facultative, anaerobic, non- 
motile coccus, forming tetrads. Does not form spores ; no lique- 
faction of gelatin occurs. 

Gelatin Stab, 22° C. — A well marked convex yellowish viscous 
mass appears on the surface in two to four days ; growth occurs 
along the needle track as white globular colonies which may become 
confluent, but generally remain distinct. No liquefaction takes 

Gelatin Streak, 22° C. — Thick grey-white layer, no liquefaction. 

Gelatin Plates, 22° C. — Lenticular, irregular and finely granular 
and irregular under microscope. 

Gelatin Shake, 22° C. — Minute colonies, discrete and globular, 

Agar Streak, 37*5° C— Well-marked viscous white layer ; colonies 
may remain distinct. 

Blood Serum, 37*5° C. — Similar to agar, no liquefaction. 

Potato, 22° C. — Well-marked white layer in forty-eight hours, 

Litmus Milk, 37 "5° C. — No coagulation of casein. Slight acid 

Broth, 37'5° C. — Thick stringy viscous deposit and general 

Pathogenesis. — White mice particularly susceptible, a minute 
quantity of pure culture causing fatal septicaemia ; the organism may 
be recovered from the heart blood, spleen, liver and other organs. 
The tetra-cocci are well marked in the tissues. Guinea-pigs are 
more resistant and generally only develop a local abscess. House 
mice, field mice and dogs are immune. 

The organism probably hastens the tissue necrosis in pulmonary 


The Klebs-Loefner bacillus occurring typically in the membranous 
exudation of faucial diphtheria may also be found in the anterior 
part of the buccal cavity, and frequently in individuals who exhibit 
no clinical or pathological signs of the disease the organisms have 


been observed in a fully virulent condition. Thus Aaser 1 found the 
diphtheria bacillus present in 17 out of 895 soldiers in a cavalry 
regiment. Park and Beebe 2 found that of 330 persons examined at 
random, 8 had fully virulent bacilli and 24 characteristic but non- 
virulent bacilli in their throats. Meade Bolton, 3 among 214 persons 
more or less exposed to the disease, found virulent bacilli present in 
41-5 per cent., and the literature teems with similar cases. 

It by no means follows that all the persons in whose throats the 
diphtheria bacilli are found are suffering at that moment from clinical 
diphtheria. In a large school of 800 children during an epidemic of 
sore throat and clinical diphtheria 4 I found 33 per cent, of the whole 
school had characteristic bacilli present in their throats, while only 
14 out of the total number of children examined showed clinical 
symptoms of the disease. In three of the cases in which no 
clinical symptoms had at any time manifested themselves, the 
organisms were in a fully virulent condition, causing the death of 
injected guinea-pigs in forty-eight hours, with all the characters 
of infection with the diphtheria bacilli. The importance of those 
ceco-parasites lies in the ease with which they may be transferred 
from one mouth to another until a susceptible individual becomes 
the recipient, when grave, often fatal, disease may result; it is, 
moreover, these " bacillustragende " persons who may come under 
the care of the dental surgeon and form an unrecognised centre of 

Occurrence. — The Klebs-Lcetrler bacilli are found most frequently 
upon the throats of persons suffering from faucial diphtheria, but are 
also found occasionally in open wounds, causing wound diphtheria, 
and upon the conjunctiva. 

The bacilli have also been found in milk, which is an excellent 
medium for their development, several epidemics having been traced 
to contamination of the milk supply from infected persons. 

The organisms rapidly die when introduced into water, and they 
have never been found in samples of water submitted to examination, 
nor have they been found in sewage, or in drain and sewer air, or 
in the emanations of decomposing animal or vegetable matter. 

The bacilli will withstand drying for several weeks, and may 
undoubtedly remain in the dust of rooms in a virulent condition. 

1 Dcutsch. Med. Woch., 1895, p. 357. - New York Med. Record, xlvi., 1894. 

3 Med. and Surg. Reporter, lxxiv., p. 799. 

4 Trans. Epidem., 1900, p. 99. 


They are easily destroyed by the action of germicides, and by a 
temperature of 58° C. for ten minutes. 

When grown in a current of air, Fernbach found that the growth 
was more luxuriant, but the life cycle shortened. The organism will 
also grow when air is entirely excluded, and is therefore aerobic and 
facultative anaerobic. The bacilli may be cultivated upon the 
ordinary laboratory media, but are morphologically most typical upon 
coagulated blood serum, the medium largely used for diagnosis. 

Lceffler's blood serum gives even better results; this medium 
consists of blood serum (liquid) 3 parts, glucose (1 per cent.) broth 
1 part. 

Coagulation and sterilization are carried out as for ordinary 
blood serum. 

Diagnosis. — In the routine examination of suspected throats for 
diphtheria bacilli, now largely carried out at the public expense in 
most British towns, a "sterilized swab " (consisting of a wire, the 
end of which is wrapped round with cotton wool, and kept till use in a 
sterilized and cotton wool plugged tube) is introduced into the throat 
and the surface touched with the sterile wool of the swab. Blood 
serum tubes are then inoculated, incubated and the culture examined 
in eighteen to twenty-four hours, when the typical bacilli are sought 
for. If diphtheria bacilli be present, the colonies, at the end of 
twenty-four hours will be larger and more denned than those of 
other bacteria present. 

Ohlmacher recommends the examination of the culture in five 
hours, even though no growth be visible, but a negative result by 
this means would hardly be of sufficient value to obviate a further 
examination at twenty-four hours ; still a positive result obtained at 
this time (five hours) is certainly of value. Equally good results 
may be obtained by examining coverslip preparations of the throat 
membrane direct and by this method the cultural diagnosis may be 
forestalled in about 30 per cent, of cases, the stain used being 
Neisser's two solutions. 

Varieties. — Two distinct varieties of the diphtheria bacillus are 
known, one the short variety, usually considered the least pathogenic, 
and the "long variety" or most virulent; the ends of the long 
variety are more frequently swollen and clubbed than the short 
variety. Both varieties form very short rods upon agar, whilst 
upon blood serum the "long" variety grows out into rods of 5 to 
8 ix in length, or even longer. 


Fig. 39. — Diphtheria Bacillus. 
Twenty-four hours' agar cultivation. Stained Gram. x 1000. (From 
Washbourn and Goodall's " Infectious Diseases.") 

Fig. 40. — Bacillus Diphtheria. 
Forty-eight hours' blood serum cultivation. Stained Gram. x 1000. 


Morphology . — Straight and slightly curved rods 3 to 4 /a long 
(twenty-four hours blood serum cultivation), often showing segmen- 
tation of the cell plasm by which the bacilli stain irregularly, espe- 
cially with methylene blue. 

By Neisser's method these bands are stained as blue dots, the 
rest of the organism brown. The bacillus usually lie grouped to- 
gether with their long axes parallel, constantly the bacilli are of 
tapering wedge-shaped form, with the bases in apposition. 

In older cultivations the ends of the bacilli become swollen and 
club shaped, forming the characteristic form. Various involution 
forms occur, the organisms becoming very much swollen, wedge 
shape, ovoid, &c. The segmentation of the cell plasm is usually 
well marked. In these older cultures red granules are often to be 
seen in specimens stained with carbol methylene blue. The organism 
retains the stain of Gram's method. 

Various branched forms have been observed in old cultures as 
has been also observed with the tubercle bacillus, it is therefore 
suggested by Hueppe and others that these two organisms are 
really only a phase in the life cycle of some higher organism allied 
to the Streptotrichese, such as S. actinomyces. Chester calls them 

The diphtheria bacillus is not known to produce spores, 
although the condensation of the protoplasm and plasmolysis often 
gives the appearance of sporulation; still the death of the or- 
ganism at the low temperature of 58° C. precludes the presence of 
true endogenous spores. 

The diphtheria bacillus is not motile and is not known to 
possess flagella. 

Biology. — Growth occurs on the ordinary laboratory media at 
37*5° G., and at 22°, the optimum temperature being that of the 
body. The organism is facultative anaerobic and does not liquefy 
gelatin, and produces no pigment. 

Gelatin Streak, 22° C. — In three days small discrete, raised 
white colonies, or confluent streak, edge indentate, no liquefaction. 

Gelatin Stab, 22° C. — Minute granular, discrete colonies to 
bottom of stab, no liquefaction. 

Gelatin Plates, 22° C. — Minute white points, granular, irregular, 
under the f" granular, irregular and yellowish-brown. 

Agar Streak, 37'5° C. — In twenty-four hours does not grow 
luxuriantly at first, but does so after several transplantations. 


Glycerine Agar, 37-5° C. — Delicate moist white to yellowish. 
Colonies. — Macroscopical (a) Superficial, delicate, grey-white, 
(b) Deep, oval, grey, entire, amor- 
Microscopical (a) Superficial, round, entire, yel- 
lowish, translucent. 

Blood Serum, 37 -5 3 C. — Opaque white or grey raised colonies, 
or dull granular moist grey streak. 

Potato, 22° C. — Glistening growth on alkaline potatoes which 
has the same colour as the medium. No growth on acid potato. 

Litmus Milk, 37'5 3 C. — Twenty-four to forty-eight hours acid, 
no coagulation, later an alkaline reaction appears. 

Broth, 27*5° C. — Twenty-four hours granular deposit with fine 
flocculi, often forming a surface film. Keaction at first acid, later 

Glucose Broth, 37*5° C. — Acid production. Acid is also formed 
from glycerine. 

Peptone Water, 37 - 5° C. — Indol produced in seven days. In old 
cultures some nitrite is also formed, so that a cholera red reaction 
is given with pure sulphuric acid (nitrate free). A slight amount 
of sulphuretted hydrogen may be produced. 

Pathogenesis. — Inoculation of animals by the subcutaneous 
method with small quantities of the diphtheria bacilli causes death 
in from three to six days. Guinea-pigs are the most susceptible, 
rabbits being considerably more resistant. Subcutaneous inoculatiou 
of guinea-pigs with 0-1 to 03 cubic centimetres of broth culture 
results in death. The pathological changes observed at the autopsy 
are extensive ecchymosis and local oedema at the seat of inoculation, 
increase of fluid in the various serous cavities, pericardial, pleural 
and peritoneal ; injected, enlarged and haemorrhagic suprarenal 
capsules, with occasionally a slight swelling of liver and spleen. 
There may be a good deal of lymphatic enlargement and congestion, 
but it is not a constant symptom. Small dotted areas of necrosis 
and fatty degeneration are often found in the liver, kidney, and 
heart muscle, more particularly in those cases in which death is 
long delayed. The most typical lesions are the fibrous-gelatinous 
exudation at the seat of inoculation from which the diphtheria 
bacillus can be recovered in pure culture, and the haemorrhagic 
suprarenal bodies. 



Fig. 41.— Bacillus Diphtheria. 
Blood serum cultivation at thirty-six hours. (From Curtis' " Essentials 
of Practical Bacteriology.") 


The bacillus is not found in the blood or in any of the organs. 

Roux and Yersin, who performed a large number of experimental 
inoculations in demonstrating the undeniable relation of the diph- 
theria bacillus to the disease of that name, found that rabbits, 
if inoculated subcutaneously with 2 cc. of virulent broth culture 
generally died in twenty to twenty- five days. Those animals which 
remained alive the longest often exhibited paralysis of the hind 
limbs, and other symptoms recalling the post-diphtherial paralysis 
of the human subject. 

Pigeons generally recovered unless inoculated with 0'5 cc. or 
more of the broth culture. Rats and mice will withstand large 
doses and are practically immune. 

Toxine formation. — It is clear that a disease such as diphtheria, 
in which widespread pathological changes are followed by the 
injection of bacilli, and yet the organisms injected are only to be 
found subsequently at the site of inoculation, must owe its symptoms 
to a poison produced by the organisms rather than to the presence 
of the organisms themselves. That this is the case was first 
demonstrated by Roux and Yersin, who filtered broth cultivations 
of the diphtheria bacillus through porous porcelain. The fluid thus 
obtained is entirely free from bacteria, but contains any soluble 
poisons produced by the activity of the organism. It was found 
that the filtered culture, when injected into guinea-pigs and rabbits, 
produced all those symptoms described as caused by the injection 
of the bacilli themselves ; no bacteria were found, however, at the 
site of inoculation, although the suprarenal glands showed the 
typical haemorrhagic symptoms. The diphtheria bacillus therefore 
produces a poison or toxine. 

The formation of the toxine goes on in the broth culture under 
certain conditions, an alkaline reaction favouring its production, 
little developing when the reaction is acid. The toxine is 
destroyed by exposure to the temperature of boiling water, and is 
much reduced in potency, although not actually destroyed, by a 
temperature of 5S° C. for two hours. It is precipitated from the 
broth culture by the addition of three or four volumes of absolute 
alcohol to one of culture, and the white precipitate thrown down 
is soluble in distilled water. When injected it produces the same 
symptoms as the injection of broth culture. 

Sidney Martin has described a method of obtaining the toxine 
in quantity, using a solution of alkali albumin to grow the organisms 


in, and purifying the alcoholic precipitate by repeated precipitations 
from water with absolute alcohol. No peptone was added to the 

Sidney Martin 1 confirmed the work of Eoux and Yersin regarding 
the enzymic nature of the toxine, and also that of Loefner respecting 
the precipitation by absolute alcohol. He separated two chemically 
different substances from the tissues of persons dead of diphtheria, 
the one an albumose, the other an organic acid. The intravenous 
injection of the albumose thus obtained caused paralysis, fatty 
degeneration, and nerve degeneration (Wallerian) in experimental 
animals, while the intravenous injection of the organic acid pro- 
duced similar effects in a lesser degree. Sidney Martin concluded 
that the diphtheria bacillus produced a toxic enzyme capable of 
digesting (fermenting) the body proteids and setting free an albu- 
mose and an organic acid which caused degeneration of nervous 
tissue. It is possible that the toxine differs from both these bodies, 
the precipitated albumoses only carrying down the poison mechani- 
cally. Eoux and Yersin found that an animal injected with 
increasing but sub-fatal doses of the diphtheria toxine developed 
immunity both to large doses of the toxine and to injection with the 
bacillus itself, and this method is now the one adopted in the pro- 
duction of anti-diphtherial serum. The animal, generally a horse, is 
injected with increasing doses of diphtheria toxine, the toxine being 
that produced by a specially virulent organism. When the animal 
shows no rise of temperature, or other reaction to the injection of 
large doses of toxine, it is bled from one of the neck veins (external 
jugular) and the blood received into sterile vessels, allowed to 
clot, the serum syphoned off and 0*5 per cent, carbolic added, to 
prevent the growth of moulds. 

The serum is then tested as described in the chapter on 

Variations in the Virulence of the Diphtheria Bacillus. — Eoux 
and Yersin found that when the diphtheria bacillus was grown at a 
high temperature, 39° to 40° C, attenuation of the pathogenic power 
was brought about ; especially was such the case where a current of 
air was passed over the culture. 

Where the virulence had fallen by the above treatment it was 
found that inoculation of animals with the bacilli and streptococci 
raised the virulence again. 

Keport L. G. B., 1891-2, p. 170. 


They also found that the organisms obtained from the mouth 
from time to time during convalescence from an attack of diphtheria, 
underwent a progressive diminution in virulence, and at the same 
time a change from the longer to the shorter forms occurred. 

As a rule the most virulent bacilli are to be found in the most 
severe cases, but it by no means always follows that the most fatal 
cases have the most virulent organisms present, the fatality appar- 
ently depending considerably upon the resisting power of the 
individual, as well as the toxicity of the organism. The bacilli may 
remain present in the mouth for long periods after all clinical signs 
of the disease disappear and, moreover, frequently retain con- 
siderable pathogenic power. Several cases, in which organisms 
have persisted for upwards of six months, are on record. Those 
cases in which a nasal discharge persists are generally those in 
which the bacilli remain the longest. 

Pseudo-diphtheria Bacillus (Hoffmann's bacillus). — An organism 
often found in cases of simple angina, tonsillitis and various mem- 
branous varieties of sore throat, much resembles the true diphtheria 
bacillus in its cultural peculiarities, but differs in the fact that it 
is not known to be pathogenic. Certain observers have claimed to 
have produced a virulent form of this bacillus, in other words, to 
have turned it into the true diphtheria bacillus by passage through 
the bodies of susceptible animals, but the evidence is by no means 
conclusive. Curiously enough the Hoffmann bacillus is generally to 
be found in the latter stages of diphtheria convalescence, but by 
no means always. Microscopically it is most difficult to distinguish 
from the short forms of the Klebs-Lceffler bacillus, and for this 
and other reasons many authorities are of opinion that it is only 
a non-pathogenic member of the same species, and that the mem- 
branous sore throats associated with this organism are in reality a 
mild non-toxic variety of diphtheria. 

The various forms of membranous disease occurring spon- 
taneously in the lower animals are due to other bacteria than the 
diphtheria bacillus, with the exception of the cat, which has been 
shown to develop true diphtheria. 

There are several other bacteria closely allied to the diphtheria 
bacillus in their microscopical and other characters, as the xerosis 
bacillus, and at least three varieties which have been isolated from 
milk. 1 

1 Eyre, Brit. Med. Congress, 1901. 



Tuberculosis is perhaps the most common disease of men and 
animals, it is chronic in its course in a large number of cases, and 
the inflammatory reaction by the body cells to the bacterial inva- 
sions has many of the characters of a new growth, so much so in 
fact that tuberculosis, together with syphilis, actinomycosis and 
glanders, have been classed by Virchow as " Infective Granulo- 
mata." The discovery of the tubercle bacillus by Koch in 1882, 
and fully described, some two years later, in one of the most 
classical series of researches in the history of pathology, gave an 
impetus to the study of bacteriology never since lost. 

Eecently, Koch has stated that the human tubercle bacillus and 
the organisms affecting cattle are different species and not inter- 
communicable. The relation of tuberculosis affecting birds to that 
affecting man has been the subject of considerable research, notably 
by Nocard, who has come to the conclusion that the two varieties 
are of the same stock, but the avian bacilli have been modified by 
their environment. 

It would be out of place here to enter into a discussion of all the 
various lesions of the human subject, or of animals with which the 
tubercle bacillus is found associated ; it is, however, necessary in 
passing to note the frequency of tubercular invasion of the glands, 
especially in children, and to point out that the mouth may often 
act as the portal through which the bacilli enter. It may also 
happen, in fact so far as one can judge often does happen, that the 
inflammatory conditions set up in the cervical lymphatic glands by 
the presence of carious teeth provides a point of lowered resistance 
in the form of an inflamed gland, and that tubercle bacilli circu- 
lating in the blood finding such a spot, settle down and develop, and 
once established, act as the centre of general infection. 

It is also extremely probable that the specific bacilli themselves 
make their way in from the mouth, along the tracks of engorged 
and enlarged lymphatic vessels, much in the same way that the cells 
of an epithelioma so rapidly spread in oral cancer. 

The bacilli are not by any means easy to demonstrate in all 
tubercular lesions, sometimes they are present in large numbers, at 
other times, as in the discharge from a tubercular sinus, or in the 
urine of tubercular kidney trouble, they are present only in small 
numbers and require the most careful t search to reveal their 


I have not been successful in demonstrating tubercle bacilli in 
carious dentine. 

Morphology . — Bacilli 2-5 — 3*5 ^ in length, 0*3 m thick. Longer 
forms, up to 5 /x, are met with. The rods stain irregularly, giving 
a jointed, or beaded appearance, so much so that the clear inter- 
spaces have been described as spores. 

At times brauched forms are met with, more particularly in the 
bacilli infecting birds. These branched forms have been described 
by Hueppe 1 and Fischel as indicating that the tubercle bacillus is 
really the parasitic form of an organism related to the Streptothrix 

The question of spore formation has not been satisfactorily 
settled, but the spores, if they exist, are much less resistant than 
the generality of true endospores. 

Stainmg Reactions. — The tubercle bacillus belongs to the series 
of bacteria kuown as " acid fast," that is, when stained they resist 
decolourisation, even with strong acids. The tubercle bacillus has 
an exceptionally resistant sheath, and therefore does not stain by 
the ordinary methods. The carbol-fuchsine method of Ziehl- 
Neelsen, gives the best results, and is employed as follows: The 
material to be stained, pus, sputum, centrifugalised deposit, &c, 
is smeared evenly over the coverslip and the film flamed in the 
ordinary way. The slip is then placed in the stain for five minutes, 
kept hot over a water bath. After staining the slip is well washed 
in methylated spirit, until no more colour is washed out, and then 
rinsed in water. The slip is now immersed for three seconds in 
25 per cent, sulphuric acid, rapidly washed in running water, trans- 
ferred to carbolic-methylene blue for five seconds, washed, dried 
and mounted. The bacilli are stained red, the tissue and cells 

The method is extremely simple, but requires some practice in 
the decolourising and subsequent double staining. 

The bacilli stain by Gram's method, but the plasmolysis of the 
protoplasm gives the appearance of streptococci. 

Biological Characters. — An aerobic, facultative anaerobic, non- 
motile bacillus, only growing at about 37° C. 

Exposure to steam, at 100° C, destroys the organism in two to 
five minutes (Scbill and Kocher). Exposure to a temperature of 

1 hoc. tit., p. 43. 



Fig. 42. — Bacillus Tuberculosis. 

Pure cultivation on glycerine agar. Several months old. 
' Essentials of Practical Bacteriology.") 

(From Curtis' 


6CK does not entirely destroy the organisms, whereas exposure to 
70° C. kills them at once (Yersin). 

Blood Scrum. — This medium was the one by which Koch first 
obtained cultivations. The colonies appear from the tenth to the 
fourteenth day as minute, irregular, hard, dry points, when ob- 
tained direct from tubercular lesions. In subsequent sub-cultures 
the growth develops more freely and may cover the whole surface, 
producing a grey, dull, wrinkled, dry layer. 

Glycerine Agar, first introduced by Nocard and Eoux, is a good 
medium for the tubercle bacillus. The colonies appear much earlier 
than serum cultures, but the medium is not a good one for obtaining 
cultures from tubercular material. The character of the growth is 
similar to the blood serum. 

Glycerine Broth. — Small white masses grow on the surface, and 
gradually fall to the bottom. If the growth be started upon the 
surface of the fluid, it gradually covers the entire surface with a 
wrinkled layer. This method is especially suitable for the produc- 
tion of tuberculin. 

Glyccr incited Potato. — A wrinkled membrane similar to the agar 
and blood serum tubes, forms on the surface of the potato ; growth 
will take place as low as 23° C. (Sander). 

Pathogenesis. — Subcutaneous injection into animals of pure cul- 
tivations, or material containing the bacilli, produces a local swelling 
in about ten days, and later breaks down, producing a deep cavity 
with caseous walls. The lymphatics leading from the site of inocula- 
tion are enlarged and the lymphatic glands later become caseous. 
Death may occur in six weeks or be delayed for two or three 
months. Instead of remaining localised to the lymphatic glands in 
the immediate neighbourhood of the injection, the bacilli may invade 
the whole body, setting up a generalised tuberculosis ; or deposits 
may occur in various organs which caseate and break down. The 
bacilli are to be found in all the lesions if careful search is made. 
In diagnostic inoculation, if the guinea-pig injected does not die 
in six weeks, it is killed and a careful bacteriological examination 
made of the lymphatics draining the inoculated region. Intravenous 
inoculation produces generalised tuberculosis, intraperitoneal injec- 
tion tubercular deposits on the peritoneal surface, and in the retro- 
peritoneal and other lymphatic glands. The spleen is commonly 
affected. Animals may be also infected by causing them to inhale 
tuberculous dust, and also by feeding with tuberculous material. 


Tubercle is common in pigs, cattle, dogs, carnivora, &c. Sheep and 
goats are practically immune. 

Tissue Reaction. — The typical microscopical change produced in 
invasion by the tubercle bacillus is the tubercle. It consists of a 
central large cell or giant cell, in and around which are to be found the 
infecting organisms ; surrounding this is an area composed of fairly 
large spindle-shaped cells or epitheloid cells, and outside these 
again a zone of unnucleated leucocytes. 

Action of Dead Tubercle Bacilli. — Prudden 1 and Hodenphyl have 
found that the injection of sterilised cultures produces tubercles, but 
that these do not result in a generalised infection, nor do the glands 
show the presence of the tubercle bacilli. The action is apparently 
due to the intra-cellular toxines held in the bacterial protoplasm, 
especially as Stockman has found that animals injected with dead 
tubercle bacilli give the tuberculin reaction. 

Koch's Tuberculin. — Koch found that when a guinea-pig suffering 
from a tubercular lesion was inoculated in another part of the body 
with dead cultures of the same bacillus, the caseating gland under- 
went ulceration and healed up and the animal did not die of tuber- 
culosis. Koch made a number of experiments which culminated 
in his attempting the treatment of tuberculosis by the injection of 
glycerine broth cultures of the bacillus in which all organisms had 
been killed by heating. 

Tuberculin is prepared by growing the bacillus in flasks of a 
broth containing 4 per cent, of glycerine, at the end of four or six 
weeks the growth has ceased and the contents of the flasks are 
collected, evaporated over a water bath to one-tenth the volume, and 
filtered through a Pasteur - Chamberland filter. The filtrate is 
crude tuberculin. 

As it was found that tubercular individuals developed so great a 
reaction to the tuberculin, and that the disease was accelerated rather 
than inhibited, the use of tuberculin as a therapeutic agent has been 
discontinued, but as a means of diagnosis in cattle and horses is 
largely adopted. 

The suspected animal is injected with the tuberculin, and if there 
be any latent tuberculosis develops a severe reaction, with rise of 
temperature, &c. According to Bang the error does not exceed 3 -3 
per cent, of infected animals inoculated. 

1 McFarland's "Pathogenic Bacteria," p. 226. 


Antiseptics. — A 5 per cent, solution of carbolic destroys tubercle 
bacilli in thirty seconds (Yersin). The exposure required in the 
presence of albuminous material is much longer. 

Light. — The tubercle bacillus is destroyed by insolation in a few 
moments when in a thin layer, and by diffused light in five days 

Immunisation. — MacFadyean 1 has recently succeeded in produc- 
ing a considerable degree of immunity in cattle by the repeated 
injection of tuberculin, followed by the injection of living tubercle 
bacilli. No anti-tuberculous serum has yet been prepared. 


This bacillus, discovered by Friedliinder in 1883, in pneumonic 
sputum, was thought by its discoverer to be the organism causing 
pneumonia. The role played by the pneumococcus of Frankel and 
Weichelsbaum in pneumonia has, however, been conclusively estab- 
lished, and the B. Friedlander is generally considered as an adven- 
titious bacterium. It often occurs in the lungs in other pathological 
conditions than croupous pneumonia. It is only slightly pathogenic 
for animals. 

Morphology. — A short bacillus, often so short as to resemble 
a coccus, generally occurring in chains of four, or in pairs. A distinct 
capsule is generally present in specimens obtained from the sputum 
direct, this capsule is similar to that observed in the pneumococcus, 
and may be stained in the same manner. The capsule often encloses 
four elements, giving the appearance of a single rod, especially when 
deeply stained. 

Staining Reaction. — Stains with the ordinary aniline dyes, but 
decolourises when stained by Gram's method. 

Biological Characters. — Aerobic, facultative anaerobic, non-motile 
bacillus. Does not form spores, and does not possess flagella. 
Gelatin is not liquefied — forms gas and ferments carbohydrates. 

Gelatin Stab, 22° C. — White, well marked growth, shining raised 
convex edge, regular on surface, along the stab well marked growth 
of white colonies, beaded, the whole described by Friedlander as 
"nail-shaped." No liquefaction occurs. Bubbles of gas are often 
found along the line of puncture. 

Gelatin Streak, 22° C. — Eaised, shining white heavy growth, no 

1 Trans. Path. Soc, Jan., 1902. 



Fig. 43. — Bacillus Friedlander. 

Gelatin stab, showing nail growth and gas bubbles. 
' Essentials of Practical Bacteriology.") 

(From Curtis' 


liquefaction. Growth rather viscid. Deep, oval or round, entire, 
brownish opaque. Surface — round convex white. 

Gelatin Plates, 22° C. — Microscopically, round, entire, brown or 
yellow, opaque with transparent borders. 

Agar Streak, 35-5° C. — White glistening, heavy, viscous growth, 

Blood Serum, 37'5° C. — Similar to agar, very viscid. 

Potato, 22 C. — Well marked, quickly covering the whole surface 
with viscid, shining whitish-yellow layer, gas bubbles are often seen 
in the growth. 

Broth, 37-5° C— Turbid, with slimy sediment, Indol slight, H,S. 

Litmus, Milk. — Well marked acid reaction in forty- eight hours, 
milk clotted in seven to ten days. 

Carbohydrates. — Dextrose, maltose, lactose, dextrin, &c, fer- 
mented with fermentation of ethyl alcohol, various acids (lactic and 
acetic) and carbonic acid gas and hydrogen. 

Thermal Death Point, 58° C. — (Sternberg). Optimum temperature 
37-5° C, but grows at 16° C. 

Pathogenesis. — Variable : rabbits are not affected, guinea-pigs 
slightly. In Friedliinder's original experiments, one dog out of five 
injected, six guinea-pigs out of eleven, and the mice (thirty- two) 
succumbed to intra- thoracic inoculation, with cultures suspended in 
distilled water. 

(9) BACILLUS INFLUENZAE (Pfeiffer's Bacillus). 
This organism occurs in the bronchial secretions and in the blood 
of persons suffering from influenza. It has so far not been found 
in other diseased conditions. It is often found in the pneumonic 
lung of persons dying from that form of influenza, and has quite 
recently been described as present in certain forms of suppuration 
following an attack of the disease, as well as in empyaema following 
influenza. The organism is extremely difficult to cultivate, and 
rapidly dies out on the culture media used. Canon, who described 
the organism simultaneously with Pfeiffer, demonstrated its presence 
in the blood of influenza patients in the following way : — The film 
is fixed in absolute alcohol for five minutes and then transferred 
to Czenzynke's stain. 

Concentrated aqueous solution of methylene blue . . . . 40 
0*5 per cent, solution of eosin in 70 per cent, alcohol . . . . 20 
Distilled Water 40 


The films are stained in this solution for three to six hours, 
and kept in the incubator (hot) during the process, after which 
they are washed, dried, and mounted in Canada balsam. 

The erothrocytes are stained red, leucocytes blue, and the bacilli 

The organisms are sometimes found in masses, but as a rule 
a prolonged search is necessary before they are found, and then 
often in only small numbers. Pfeiffer was, however, unable to 
confirm the presence of the organisms in the blood. 

Morphology. — Small bacilli 05> long, by 0-2 ^ wide ; solitary, 
or united in pairs, and occasionally in chains of three or four. 

Staining Beactions. — The bacilli stain badly with the ordinary 
stains, best with carbol-fuchsine dilute, or with Lcefner's alkaline 
methylene blue, or Czenzynke's stain. 

Polar staining is generally well marked, the bacilli giving the 
appearance of diplococci. 

They do not stain by Gram's method. 

Biological Characters. — An aerobic (facultative anaerobic ?), non- 
motile bacillus. Not known to form spores. It does not grow upon 

Glycerine -agar appears to be the only medium upon which the 
organism grows at all well, and even upon such a medium the 
development is scanty, and often requires a lens to demonstrate 
its presence. 

The colonies are very small, transparent, and difficult to see. 

According to Kitasato, the colonies always remain separate from 
one another, and do not coalesce as the majority of organisms do. 
This is considered typical and diagnostic of the organism (Kitasato). 

Broth. — A small amount of growth occurs at the surface and 
gradually sinks, producing a slight woolly deposit. 

The bacillus rapidly dies when dried and succumbs to a tem- 
perature of 60° (Pfeiffer). 

The optimum temperature is that of the body. 

Pathogenesis. — Eabbits and guinea-pigs often die when injected 
intravenously with cultures of the influenza bacillus, the chief 
symptoms, according to Pfeiffer, being a great rise of temperature, 
with subsequent paralysis of the hind limbs. Deline and Kole found 
that they could not produce immunity by the inoculation of gradually 
increasing doses of the bacillus, as is the case with many other 
bacteria (cf. diphtheria). A certain amount of temporary immunity 


resulted in Deline and Kole's experiment, but the animals were 
never capable of resisting large doses, and no true resistance was 

This experimental fact is interesting in relation to the clinical 
fact of the absence of protection afforded by an attack of the disease, 
the susceptibility to a second attack being rather increased. 

It is probable, however, that after a large number of attacks 
some slight immunity may be set up, as the disease appears to 
gradually become less and less severe in type. 

(10) B. PYOC YANK US. 
(Pseudomonas Pyocyanea Migula.) 

This organism is found in abscesses when the contents are of 
the peculiar bluish or green colour known as " blue pus." 

It occurs occasionally in the mouth and throat, and may be 
found at times in the cavities of tubercular lungs, in otitis media, 
meningitis, &c. It is not uncommon in dust. On agar cultures 
especially this organism produces well marked crystals. One of the 
most characteristic appearances associated with this organism is the 
green-blue pigment that is formed on most media. Gessard has 
shown that this pigment is composed of at least two different bodies 
(fluorescin and pyocyanin), and that by modifying the conditions 
under which the organism is grown it may be made to produce one 
or other at will. 

Hoiyliology. — Slender bacilli 1-5 to 2 m in length, and 0-25 to Oo 
a* in thickness ; at times two or more may be found jointed together. 
The rods are actively motile, and possess flagella. No spores are 

Staining Reactions. — Stains by the ordinary aniline dyes, and 
retains the stain of Gram's method. 

Biological Characters. — Aerobic, facultative, anaerobic, motile, 
liquefying bacillus. Forms pigment. No spores formed. Pathogenic 
for animals. 

Gelatin Stab, 22° C. — In twenty-four to thirty- six hours a faint 
grey line appears along the needle track, and at forty-eight to seventy- 
two hours liquefaction commences, and a slight green tint appears 
in the upper part of the gelatin. The liquefaction proceeds in cup- 
like form, and a green colour at the same time diffuses through the 
gelatin. The liquefied gelatin is cloudy and shows a deposit in the 
deeper parts. 


Gelatin Streak, 22° C. — A groove of liquefaction is produced in 
forty-eight hours, the medium becoming gradually tinted green. 

Gelatin Plates, 22° C- — Small whitish points appear, which under 
the microscope are brownish-yellow with nodular surface, and sur- 
rounded with a sphere of liquefaction in the deep layers. On the 
surface the colonies are flat, edge entire, surface reticulated as 
seen under the microscope, and surrounded with shallow cups of 
liquefaction. A fine haze of greenish colour appears round each 
colony, rapidly permeating the whole plate. By about the fifth day 
the plate is entirely liquefied. 

Fig. 44. Bacillus Pyocyaneus. 
Twenty-four hours' agar cultivation, x 1000. 

Agar Streak, 37"5° C— In twenty-four hours a well-marked moist 
white layer is formed, and slight tinting of the medium has occurred. 
In forty-eight hours the colour is bright green due to fluorescin, 
which is soluble and so diffuses through the medium. Later the 
green colour changes to an olive green or reddish-brown from the 
development of a second pigment, pyocyanin, which requires 
the presence of peptone for its formation, and is insoluble. Upon 
fresh agar cultures well marked crystals are seen along the line of 


Potato. — Luxuriant growth, at first green, later turning brown, 
both at 22° and 37-5° C. 

Blood Scrum, 37*5° C. — Similar to agar, but liquefaction occurs 
in a well-marked groove. 

Litmus Milk, 37 -5° C. — Well marked coagulation of casein, which 
later is dissolved and ammonia given off. 

Broth, 37*5° C. — Well marked fluorescence and general turbidity. 
A scum, often a distinct pellicle, is formed, and a thick precipitate 
collects at the bottom of the tube. Both pigments are produced ; 
the fluorescin being soluble in chloroform may be separated for 
the insoluble pyocyanin. 

Peptone Water. — With the addition of 5 per cent, glycerine the 
blue pigment pyocyanin only is formed. No indol formed. 

Egg Albumin. — The green pigment fluorescin alone is formed. 
It is soluble in chloroform, and crystallises out as long needles. 
On the addition of weak acid the colour changes to red. 

Glucose Broth. — Acid, no gas. Nitrates reduced to nitrites. 

Pathogenesis. — One cubic centimetre of broth culture injected 
intraperitoneally or subcutaneously generally causes death in rabbits 
and guinea-pigs in twenty-four to thirty-six hours. At the autopsy 
inflammatory oedema and infiltration, sometimes a well-defined 
abscess, are found at the seat of inoculation. The peritoneal cavity 
shows a similar fibrinous inflammation when the organism is injected 
into that cavity. The organisms may be found in small numbers in 
the blood and various organs. 

Intravenous injections generally produce rapidly fatal septi- 
caemia with nephritis, occasionally chronic wasting accompanied 
with albuminuria. Immunity may be produced by the injection of 
gradually increasing doses, commencing with a sub-fatal dose. The 
animals thus immunised show a decidedly increased resistance to 
infection by the anthrax bacillus. Woodhead and Wood also found 
that the injection of sterilised cultures of B. pyocyaneus directly 
following injection with anthrax bacilli protected against that 

A large number of varieties of this organism have been described, 
some of them being no doubt varieties of the B. pyocyaneus, in 
which the power of pigment production has become, as Gessard has 
shown it may, so modified that the production of either pigment 
may be prevented by alteration of the nutrient medium. The 
antagonism of the B. pyocyaneus and B. anthracis referred to above 


is interesting as an example of protection afforded by dissimilar 
diseases. The relation of the B. pyocyaneus to the tetanus bacillus- 
is of quite another order. The tetanus bacillus, under ordinary 
circumstances a strict anaerobe, will grow in broth freely exposed to 
the air if B. pyocyaneus is also present in the culture. 

There is usually little difficulty in recognising the B. pyocyaneus,. 
when obtained from pus or other material, by the peculiar pigments 


The fungus of actinomycosis was discovered in 1877 by Bollinger, 
although Langenbeck, as early as 1845, had found that the disease 
of cattle known as actinomycosis could be transmitted to man. 

The disease is almost confined to animals, for the most part 
cattle, but since attention has been directed to the disease it appears 
to be more frequent in man than was at first supposed, and a large 
number of cases are now on record. 

The organism itself belongs to the higher bacteria, and shows a 
far greater complexity of form than is exhibited by the majority of 

The point of infection is commonly the mouth, the organism 
gaining access to the tissues either through a carious tooth, or as 
the result of some slight local injury. In a number of cases 
inoculation has apparently taken place through the medium of an 
awn of barley containing the fungus, which has become imbedded in 
the soft tissues. It is common to find these grains imbedded in the 
local lesion. Two chief varieties of the disease are known, the one 
in which considerable local reaction with enlargement and thicken- 
ing of the tissues and bone occurs, the other a condition of general 
infection with deposits and abscesses in various organs, notably the 
liver. The chronic form with local swelling was for a long time 
confounded with osteo-sarcoma. In the pus from the abscesses or 
local lesion small yellowish-grey granules are to be seen even with 
the naked eye. Microscopically these granules show the peculiar 
rosette-shaped fungoid masses, consisting of a central mass sur- 
rounded by threads which give the rayed appearance to which the 
fungus owes its name (Hertz). The granules are each composed of 
a central mass of cocci-like bodies (gonidia) often containing a 
quantity of dark pigment. Surrounding the central portion is a 
zone of tangled threads showing true branching, generally lateral. 


The ends of the threads are commonly but not invariably clubbed, 
and at the periphery of the granule give the appearance of rays. 
The threads are about 0'5 m in diameter, and are composed of a 
central protoplasmic axis surrounded by a gelatinous sheath. In 
young specimens the threads take up aniline dyes uniformly, but in 
old cultures the threads tend to stain irregularly and may appear 
as chains of cocci or bacilli. The clubs do not stain by Gram. 
In culture media the changes are somewhat different. The 
branched and tangled mass of threads are formed as colonies of 
cartilaginous consistency ; the clubbing is not marked. The 
threads stain irregularly after about a week, and the gonidia are 
well marked, often covering the colony with a white cr yellow 
dusty efflorescence. The typical granules are not formed, although 
there may be some attempt on liquid blood serum. 

The conditions of growth outside the body and the form in 
which the organism exists when infection occurs is not at present 
known. It is thought that it may exist in the grains of certain 
cereals in a similar manner to Puccinia graminis. 

Morphology. — Filamentous, branched, and club-shaped forms 
(fig. 1, i.), with all the morphological forms of bacteria represented 
at times. The clubs are not formed in cultures. Various changes 
occur in the threads, which at first stain well but later become 
granular and stain irregularly. No endospores are formed, but 
gonidia are present. 

Staining Reactions. — Stains with the ordinary aniline dyes, best 
by Gram's method, w ? hich is much the best stain for tissue prepara- 
tions. The clubs do not retain the stain of Gram's method, but may 
be counterstained with picric acid. 

Biological Characters. — An aerobic facultative anaerobic strepto- 
thrix, forming gonidia, non-motile; does not possess flagella. 
Gelatin is liquefied. 

Gelatin. — The organism grows slowly at the room temperature, 
and the medium is gradually liquefied and turns a dark brown 
colour, the liquid being somewhat viscous. Scattered about in the 
fluid are small round white nodules, from which filaments radiate. 

Agar and Glycerine Agar. — After three days at 37 - 5° C., minute, 
hard, spherical, white colonies appear (fig. 45, a) ; these gradually 
increase, and become raised at their edges, ultimately forming an 
undulating and crater-form surface, at first yellowish, later greenish- 
grey (fig. 45, b). The older colonies often resemble lichen (fig. 45, c), 


A B G 

Fig. 45. — Streptothrix Actinomyces Cultivations on Glycerine Agar. 
A. Discrete rounded colonies, after about ten days' incubation at 37° C. 
B. Limpet-shaped colonies three and a half months old. C. Lichen-like 
appearance frequently seen ; the growth is three and a half months old. (From 
Curtis' "Essentials of Practical Bacteriology."), 


and have a yellowish or ashen-grey tint. The corrugated surface 
is covered with a powdery dusty layer. The colonies are extremely 
difficult to remove for examination. 

Potato. — Similar appearance to agar, but more luxuriant growth. 
The colonies are quite unlike those produced by other bacteria, the 
streptothrix of madura-foot being the only organism at all resem- 
bling them, and this organism colours the potato a dark red. 

Pathogenesis. — Intra-peritoneal injections of the bacillary or 
filamentous form of the parasite in rabbits and guinea-pigs is 
followed in about a month by nodule formation. The nodules, 
composed of granulation tissue (granuloma), are vascular on the 
surface, and contain curdy pus, in which the typical colonies are 
found. In man the disease may take one or both of the forms 
noted above. Sometimes large areas of bone become carious and 
necrosed, the disease being classed by Virchow with glanders and 
tubercle as infective granulomata. Infection of the bowel may 
occur, ulceration and extensive necrosis following. The organism 
has also been described in the ovaries and fallopian tubes (Muir 
and Granger Stewart) ; it has also been found in the brain, liver, 
spleen, &c. In the later stages of the disease deposits may occur 
in the various organs with the formation of metastatic abscesses 
containing the typical colonies. The diagnosis is easy, both by the 
typical granules in the pus of the abscesses or other lesion, and 
the characteristic growth on agar and potato. 


Found by Miller in unhealthy mouths and along the gum 
margin in such cases. I have also observed this organism in several 
cases both of dental caries and in gingival inflammation, and 
have therefore worked out the biological characters, as those given 
by Miller only include growth on gelatin and agar. 

Morphology. — Bacilli from 2 to 6 m long, 0-5 to 0*75 m wide, 
often jointed in pairs or in chains. The elements may at times be 
curved. Ends square or rounded. Two or three bacilli may at 
times lie side by side somewhat in the manner of the Klebs-Lceffler 
bacilli. Involution forms (globular or twisted) are common on old 

Staining Reactions. — Stains by the ordinary aniline dyes and by 
Gram's method. The flagella may be stained by Pitfield's method. 

Biological Characters. — An aerobic facultative anaerobic, liquefy- 


ing, motile, chromogenic bacillus. Forms spores which resist a 
temperature of 75° C. for half an hour. Grows in the usual culture 
media, best at 37-5° C. 

Gelatin Plates, 22° C. — In forty-eight hours irregular spreading, 
raised colonies, with irregular and wavy edge, yellow centre lying 
above the outer paler mass of colony. In three days the colonies 
liquefy the gelatin and float upon the surface of liquid as round 
crinkled masses with thickened centre, which is now yellow-brown 
in colour. The gelatin becomes dark brown. 

Gelatin Stab, 22° 0. — Forty-eight hours, growth to bottom of 
stab; three days, slight cup-shaped liquefaction which gradually 
approaches the walls of the tube. In newly isolated cultures the 
cone may remain empty. White flocculi form in the fluid later. 

Gelatin Shake, 22° C. — Forty-eight hours, cloud of minute 
colonies but no gas bubbles. Liquefaction commences at the sur- 
face, and does not take place in the depths. 

Gelatin Streak, 22° G. — Forty-eight hours, slight liquefied groove 
with little other evidence of growth ; four days, the fluid becomes 
filled with yellowish-white flocculi with radiating processes. 

Agar, 37 # 5° C. — Twenty-four hours, heavy growth, with tendency 
to spread at intervals along the streak in club-shaped processes 
(lobulate) ; the central portion of the streak and of the club-shaped 
processes is buff-yellow, the edge grey-white. The medium may be 
tinted brown. 

Blood Serum, 37*5° C. — In twenty-four hours a broad, deep 
groove of liquefaction is formed with brown discoloration of medium. 

Potato, 37'5° G. — Twenty-four hours, well-marked, dry, yellow- 
brown growth, granular aud glistening ; the potato becomes coloured 

At 22° C. a good deal of yellow pigment is formed. 

Broth, 37*5° C. — Twenty-four hours, faint growth of isolated 
flocculi iu fluid ; no general turbidity. The flocculi sink and form 
a thin deposit in three or four days. Good indol reaction in seven 
days. H S. scanty. 

Litmus Milk, 37-5° C. — Forty-eight hours, well-marked acidity 
with coagulation which does not become dissolved for two or three 
days. No gas is formed, and no smell given off. 

Glucose broth, lactose broth, maltose broth — acid fermentation 
in forty-eight hours. 

Seven days' agar culture suspended in broth and heated to 


75° C. for half an hour gives a good culture on sub-cultivations 
being made. 

The spores, which are small, stain by Mollers' method. 

Pathogenesis (Miller). — Pathogenic for mice, rabbits, and guinea- 
pigs when intravenously injected in doses of 0-25 cc. of a 
broth culture. At the autopsy peritonitis, sometimes purulent, 
is observed. Death occurs in ten to twenty-four hours. Sub- 
cutaneous inoculation resulted in local abscess only. 

(13) B. GANGRiENJE PULP^E (Arkovy). 
B. mesentericus niger (fuse us). 

Found by Arkovy in dead tooth pulps, in carious dentine, and 
in the oral secretions. 

Morphology. — Bacilli about 4 /* in length, with rounded ends 
(not sharply denned as first stated, and not pleomorphic) ; often 
pairs are united at an acute angle. 

Staining Reaction. — Stains with the ordinary aniline dyes, and 
by Gram's method. The so-called cocci described by Arkovy do 
not stain by methylene blue, and are spores 1 which are large and 
oval, and may be stained by the usual methods (hot carbol- 
fuchsin, &c). 

Biological Characters. — An aerobic, facultative anaerobic, motile 
liquefying bacillus ; forms pigment and spores. 

Gelatin Plates. — In twenty-four hours minute white colonies 
make their appearance, resembling flour dust. These gradually 
become slightly yellow in colour, and in thirty hours are confluent, 
whilst the whole of the medium is liquefied with a whitish 
wrinkled pellicle covering and floating on the surface of the fluid. 
An extremely unpleasant smell is given off resembling old cheese. 

Gelatin Stab. — At the end of forty-eight hours liquefaction 
commences, and soon reaches the wall of the tube. Flocculi form 
in the fluid, and in ten days a wrinkled pellicle has formed upon 
the surface. The gelatin is coloured a red brown, the pellicle 
being of a dirty brown. The liquefied gelatin gives a strongly 
alkaline reaction. 

Agar Plates, 37'5° C. — At the end of tw T enty-four or thirty-six 
hours, small white colonies make their appearance. They have a 

1 Cent, fur BaktcrioL, Bd. xxix,, No. 19, 1901. 


flour-dust form similar to those on the gelatin plates. Occasionally 
the colonies (surface ?) are larger, flat and leaf-like, and marked 
with fine striae. The same unpleasant smell noticed in the gelatin 
plates is also present. 

Agar Streak, 37*5° C. — A wrinkled layer, five to six millimetres 
broad, is formed, which becomes brown after five or six days ; the 
medium is itself also coloured a brownish tint. 

According to Eader's international colour scale, the tints pro- 
duced on the different media are as follows : — 

(1) Agar, colour of growth on surface ... 33 brown — a 

(2) Gelatin reflected light ... ... ... 33 brown — d 

(3) Gelatin transmitted light ... ... cinnabar — 3a 

Blood Serum, 37*5° G. — A brown liquefied streak is produced 
along the needle track. 

Broth, 37-5° C. — In thirty-six to forty-eight hours a well-marked 
pellicle is formed, having the same colour as the gelatin cultivation. 

Potato, 37 # 5° C. — A moist brownish wrinkled skin forms over 
the surface, and the medium is coloured a deep brown. 

Milk, 37*5° C. — No acid is formed, but a precipitation of the 
casein occurs. 

Pathogenesis. — Subcutaneous inoculation in mice produced diar- 
rhoea and death in twelve days. The bacilli were found in the 
blood. The bacillus is also pathogenic for rabbits and guinea-pigs 
when injected in large doses subcutaneously. 


Found by Miller in the superficial layers of carious dentine. 

Morphology. — Slightly curved bacilli with pointed ends, solitary 
or in pairs. 

Biological Characters. — An aerobic, facultative anaerobic, non- 
liquefying bacillus. Spore formation, motility, staining reactions 
not given. 

Grows well in the usual culture media. 

Gelatin Plates. — Spherical colonies with concentric rings, almost 
colourless except under the microscope, when they are slightly 
yellow. The gelatin is coloured a faint green. 

Gelatin Stab. — A limited growth occurs along the track of the 
needle and a considerable growth upon the surface. No liquefaction 


Agar Streak. — A thin growth with an irregular margin occurs 
along the track of the needle. The growth is bluish by transmitted 
light and greenish-grey by reflected light. 

No other cultural characters given. 

Pathogenesis. — Injections into the peritoneal cavity of mice 
and guinea-pigs usually cause fatal peritonitis in from one to six 
days ; the bacilli are found in the blood in small numbers. Subcu- 
taneous inoculation into animals produced severe local suppuration. 


Obtained by Miller from a gangrenous tooth pulp. 

Morphology. — Bacilli slightly curved and pointed, occurring in 
chains, in pairs, or solitary. 

Biological Characters. — An aerobic, facultative anaerobic, liquefy- 
ing bacillus. Spore formation, motility, staining reactions not given. 

Gelatin Plates. — Large, darkish, yellow-brown colonies appear, 
which in eighteen hours produce liquefaction, and soon liquefy the 
whole of the gelatin. 

Gelatin Stab.— Liquefaction begins within forty-eight hours and 
gradually extends, the liquefied gelatin being separated from the 
non-liquefied portion by a horizontal plane. 

No other biological characters given. 

Pathogenesis. — Injections of 005 cc. into the peritoneal cavity 
produced death of white mice in eighteen to thirty hours. 


Obtained by Miller from a case of suppurative periodontitis 
three times at intervals of three months, and once in a very dirty 

Morphology. — Irregular cocci or plump rods, occurring solitary 
or in pairs. 

Biological Characters. — An aerobic, facultative anaerobic, non- 
liquefying micrococcus. Grows w T ell at the room temperature and 
in the usual culture media. 

Gelatin Plates. — Forms spherical, well-defined colonies, with a 
sharp margin, which at first are colourless under the microscope, 
but later become opaque. 

Gelatin Stab. — A well-marked growth occurs along the line of 
puncture and a copious raised growth on the surface. No lique- 
faction occurs. 


Agar Streak. — A thick, greyish, moist growth occurs in twenty- 
four hours, which has a purple tinge by transmitted light. 

Sugar Media (composition not stated). — A considerable develop- 
ment of gas occurs and a strongly acid reaction is soon present. 

Pathogenesis. — Subcutaneous injections into mice were followed 
by local abscess and necrosis, and sometimes by the death of the 
animal. Intraperitoneal injection invariably produced death in 
twelve to twenty-four hours. 

[Staining reactions and growth on milk, potato, broth, &c, 
not given. This organism is probably nearly related to B. coli 


Dental Caries. 

The destruction of the tissues of the teeth, commonly known as 
dental caries, is a process distinctly allied, both in its chemical and 
bacteriological aspects, to the general phenomena of putrefaction. 

Dental caries, like putrefaction, is rarely caused by one species 
of organism, and the reaction of the medium in both caries and 
putrefaction undergoes fluctuation from acid to alkali ; and in both 
processes an alternation of species takes place according to the phase 
of phenomenon. 

The disintegration of enamel, dentine, and cementum is brought 
about in the first instance by the action of various organic acids, 
mainly lactic, produced by the vital activity of bacteria ; subse- 
quently the various digestive ferments also produced by the 
organisms come into operation, dissolving the decalcified matrix 
of the cementum and dentine. What, however, occurs in the first 
stages of dental caries, and why certain races of men and the 
majority of animals appear to be immune, is a much more difficult 
matter to elucidate. 

Artificial caries, so well demonstrated by Miller, is by no 
means difficult to reproduce by exposing cubes of dentine to the 
action of micro-organisms of the acid-producing class in solutions 
containing carbohydrates. 

Caries of enamel is much more difficult to reproduce arti- 
ficially, uniform denudation of the enamel generally taking place 
in carbohydrate cultivations ; and as the process of natural caries 
generally originates at some point of the enamel surface rather than 
over the whole tooth, much interest centres around the initiation of 
the process. 

Very many theories have been advanced at various times to 
explain the commencement of enamel destruction, ill-developed 
and pathological conditions of the enamel structure, deficiency of 


lime salts — said to be associated with insufficient calcium salts in 
the water of a country — diet, civilisation, alteration of physiological 
relationship in position of the teeth in the jaw from evolutionary 
causes, &c. Many, if not all, of these factors may have their place 
in predisposing causes, but the ultimate liberating cause is often 
overlooked in the multiplication of predisposing ones. 

Bead 1 has lately advanced the proposition that caries may be 
due to the alteration in the sort of flour used in bread-making. 
Thus the finer roller-ground flour has to a large extent replaced 
the old stone-milled article ; and Bead states that the amount of 
acidity in terms of lactic acid produced by chewing bread made 
from either sample is much greater in the case of the finer roller 

Samples of bread made from the two flours were chewed for 
equal lengths of time and the acidity determined ; in every case the 
acidity of the roller flour was in excess of the stone-milled. That 
such an acidity might predispose to caries is by no means im- 
probable, but the subject is not yet sufficiently investigated to 
draw definite conclusions. 

Sim Wallace 2 considers that " the cause of the prevalence of 
dental caries is that the natural food- stuffs are to a large extent 
ridded of their accompanying fibrous parts," and that due to the 
same cause " the micro-organisms of the mouth lodge and multiply, 
and augment the rapidity and intensity of the acid fermentation." 

That such a factor does occur is certain, but it hardly explains 
the fact that animals, dogs, cats, monkeys, rabbits, &c, which 
exhibit no dental caries, are yet found to have food particles 
frequently lodged between their teeth after a meal, the fibrous 
matters themselves remaining impacted. Interstitial caries is rare 
in animals and yet commonly occurs in man. 

The researches of Miller, Mummery, and more recently Leon 
Williams, have shown that the enamel surface in sheltered positions 
is often covered with a film-like layer of bacteria, and that under 
such a layer definite disintegration of the enamel is taking place. 
Moreover many of the bacteria found in this film may be seen 
in the spaces hollowed out between the enamel prisms ; the spaces 
evidently have been formed by the action of the acids produced 
by the bacteria ; moreover spreading from these points of attack 

1 Jonm. Brit. Dent. Assoc, 1900. - " Cause of Decay in Teeth." 


and passing inwards to the dentine may be seen organisms well in 
advance of the general process. 

The acid produced by the bacteria, although in small quantities, 
would eventually inhibit the growth of the organisms were it not 
neutralised as soon as it is formed by union with the lime salts of 
the enamel, each fresh increment of acid attacking a fresh portion of 
tissue, the resulting products diffusing into the saliva. 

Miller figures several examples of this bacterial layer attached to 
the surface of enamel, and also shows the organisms permeating the 

Fig. -iG. — Dental Caries. Section of Enamel with Layer of attached 

Organisms and formation of Pits between Enamel Hods. 

Photomicrograph and specimen by Dr. Leon Williams, x 500. 

enamel substratum. Miller also showed, by a series of admirably 
conducted experiments, that the familiar appearance of caries is due 
to the various acids evolved from the carbohydrate constituents of a 
normal diet. 

Leber and Rottenstein endeavoured to produce artificial caries 
by placing normal teeth in a mixture composed of the various 
constituents of a normal diet. They failed to produce caries 
experimentally, and came to the conclusion that it was not the 
result of bacterial activity. They admitted that their experimental 


flasks, &c, gave off obnoxious smells, and that ordinary putrefaction 
occurred, in fact they abandoned their experiments largely on this 
account. Miller however by so adjusting the condition that a 
constant acid reaction was present, obtained strikingly confirmatory 
results of the role played by organisms producing acid fermentation 
in the pathological condition known as dental caries. 

Leber and Eottenstein's failure is of easy explanation, but 
although they failed in their main object their work is of consider- 
able importance in the aetiology of caries as demonstrating by a 
negative result the necessity of an acid fermentation to initiate the 

In the ordinary phenomenon of putrefaction an acid reaction may 
be present, but very soon gives place to distinct alkalinity and the 
evolution of evil-smelling gases — indol, skatol, sulphuretted hydro- 
gen, and, as a rule, ammonia. 

The phenomena of putrefaction are by no means all produced 
by a single species or race of organisms, one succeeding another as 
the conditions suitable for their development arise (see p. 21). 

But more important still is the observation of Maly, 1 who took 
the mucous membrane of the stomach, placed it in a solution of 
cane sugar and kept the mixture at the body temperature for several 
days. The lactic acid arising from the decomposition of the sugar 
was neutralised from time to time, and it was found that the process 
of acid production continued until all the carbohydrate present 
had been converted into lactate ; and then, and not till then, did 
putrefactive odours become manifest. 

This, then, fully explains the failure of Leber and Eottenstein, 
and the success of Miller's experiments. 

It will be noticed, moreover, that the acid produced by the 
organisms requires to be neutralised from time to time ; otherwise, 
owing to the increased acidity of the fluid, the organisms cease their 
activity, the very acid produced acting as a check to the growth 
of the bacteria producing it. 

In examining the statistics of the incidence of caries as occurring 
in various races of mankind, and in considering the diet of such 
people, we find that those races whose diet is mainly meat show 
a far smaller percentage of caries than do the races whose diet 
is principally carbohydrate. 

1 Herman's Handbuch, Bd. v. (2), S. 239. 


It is moreover observed that the teeth of persons suffering from 
enteric and other diseases necessitating a prolonged subsistence, 
during convalescence, on a diet composed of a considerable pro- 
portion of carbohydrate, often show an increased amount of caries 
unless most careful cleansing of the mouth is carried out by the 
attendants. Millers, bakers, and persons engaged in sweet-stuff 
factories invariably exhibit a marked amount of caries ; in millers 
and bakers particularly, marginal cavities are common. Caries is 
moreover more common in children of both sexes than in adults, 
and more frequent in adult woman than in man. All the various 
questions relating to fermentation point to food-stuff composition as 
of great importance in the aetiology of dental caries, but in applying 
this general principle we are met with several obstacles which 
apparently militate a good deal against the acceptance of the theory 
of food-stuff origin of caries. 

One of the chief and main difficulties in discussing the whole 
problem is the attempt to explain the process of dental caries with- 
out having regard to the importance of several rather than a single 
predisposing cause ; the explanations advanced by various observers 
are none of them sufficient per se to elucidate the problem in its 
entirety, but taken together a very good working hypothesis may 
be obtained. Dental caries is not a specific disease due to a 
certain specific micro-organism; it is no definite "entity," but a 
process occurring through the operation of certain biological and 
physical Laws. 

It has been suggested by some observers that a deficiency in the 
amount of lime salts of teeth may contribute to the early develop- 
ment of dental caries. 

Black, and later C. Tomes, estimated the percentage composition 
of a large number of teeth and found that there was no appreciable 
variation, and that there was no evidence that the incidence of caries 
could be associated with a decrease in the percentage of the lime 
salts present. 

It is known however from an empirical point of view that some 
teeth are apparently mere liable to the inroads of bacteria than are 
others, although the researches just cited show that the suscepti- 
bility to caries probably does not lie in the lime salt content. 

Leon Williams has demonstrated the plaques of micro-organisms 
on the enamel surface of teeth, and the incipient caries occurring 
under these plaques, and I have myself constantly observed these 


sheets ■ of bacteria in various situations on the enamel surface. 
During investigations carried on concerning the flora of carious teeth 
I have constantly met with a series of bacteria which are character- 
ised by a curious facility for forming extremely tough gelatinous 
colonies, not by any means due to the presence of carbohydrate as 
in the mucinous fermentation of sugar and molasses, but occurring 
on media free from any carbohydrate whatever ; one organism in 
particular, a coccus, is frequently present. This organism is 
frequently to be met with upon the enamel surface of teeth, par- 
ticularly the white opaque patches of softened enamel to which 
Williams has drawn attention (for description see p. 172). 

I found no difficulty in reproducing the plaque-like layer upon 
sterilised teeth suspended in a cultivation of one of these bacteria, 
and moreover when another organism capable of acid fermentation 
was mixed with the plaque-forming organism, and carbohydrate 
media used, under the plaques formed upon the enamel surface 
by the two bacteria superficial disintegration of the enamel was 
observed to occur in a week to ten days. Such an experiment is 
no doubt largely in favour of the organisms ; there is no cleansing 
due to mastication or movement of tongue or saliva, and no great 
and constant dilution of the acids formed, which are at liberty 
to attack the tooth under the bacterial sheet. Nevertheless the 
colonies, which are often formed even upon the surface of the 
glass in the culture tube, are remarkably adherent and resist 
removal, and it is not unfair to suppose that such a condition 
obtains in the mouth. An important coincidence to this supposition 
is afforded by the fine teeth of many native races, many of which, 
particularly the Zulus and Kaffirs, are particularly assiduous in 
cleansing their teeth. Amongst the former it is the common 
practice for the Zulu mother to carefully cleanse her child's mouth 
after every meal until it is old enough to do so for itself; the 
finger is generally used, and some ashes (wood) from the fire are 
employed. The majority of the adult natives in the beds of the 
Seamen's Hospitals are especially careful of their teeth, at times 
refusing to eat unless first supplied with water with which to wash 
their mouths after the meal. 

In making cultivations from the mouths of natives with good 
dentition, and also from the mouths of some of the monkeys at the 
Zoological Gardens, I have been struck with the number of putre- 
factive rather than acid-forming bacteria present in the mouths. 


Certain of these bacteria will also form a definite layer upon the 
surface of enamel when a sterilised tooth is suspended in a broth 

It is a frequently observed clinical fact that individuals applying 
for treatment at dental hospitals may possess peculiarly dirty 
mouths, with marginal inflammation of the gums, and yet exhibit 
extremely small evidence of caries ; the appearance of such 
mouths is strikingly similar to several of the monkeys I examined. 
Putrefaction was evidently the ascendant process and therefore an 
accompanying alkaline reaction, such carbohydrate food as was 
taken probably happening to be of a species only fermented with 
difficulty. The coincidence is interesting, and if open to several 
explanations is not opposed to the general principles we have 

I have already referred to the question of roller-flour, and to the 
increased amount of acid it is said to engender. Acid may be 
present in the mouth in certain pathological and physiological con- 
ditions, in pyrosis, the vomiting of pregnancy ; iu diabetes mellitus 
an acid saliva is frequently present. Acid contained in medicine 
has also to be mentioned in the same category. 

It is possible that small quantities of acid frequently applied to 
the teeth may produce microscopical irregularities, or what is more 
important, solution of the interprismatic substance of the enamel 
prisms, or of the axial portions of those prisms, assisting in the 
adherence of organisms and forming microscopical points of entrance 
from which portals the process may extend. 

On the other hand we must not forget that some people are in 
the habit of consuming acid foods, and eating strongly acid fruits, 
as is the case with the Sicilians 1 , and it is conceivable that the 
acid may act as a protective by preventing the development of acid- 
forming organisms, or dissolving away the outer layers of the 
enamel and with it the contained bacteria, secondary dentine occlud- 
ing the pulp chamber before the process had threatened that cavity. 
Such an obliteration is common in old skulls with great denudation 
of the dentine. 

So far we have not considered the relation of pathological mal- 
formations of the teeth in their relation to caries. 

1 Cosmos, 1898. Dr. Leon Williams tells me that the Sicilians, who are 
particularly free from caries, are large consumers of lemons. 


Leon Williams has pointed out that pits, grooves and fissures, 
pigmentation, granular and amorphous enamel are to be found in 
the lower animals whose teeth are comparatively free from caries, 
and that various species of human enamel, which are apparently 
especially liable to caries by reason of their pathological irregulari- 
ties, may resist for years, whereas enamel, to all appearances sound, 
is often the seat of rapid decay. To such a statement all will 
agree, but it is an undoubted clinical fact that hypoplasic teeth do 
certainly undergo extremely rapid caries under certain conditions, 
the point of attack being almost invariably a pit or fissure on the 
enamel surface. Looking at the question from a broad general 
point of view it certainly by no means follows that because a tooth 
has a developmental defect therefore it must become carious, but we 
may say with a considerable measure of truth that exposed to those 
conditions which we have seen predispose to caries, the tooth with 
irregularities and deficiencies on its enamel surface is more liable to 
attack thau the developmentally perfect one. Further, we are not 
sufficiently conversant with the microscopical defects or normal 
structure of the enamel surface of teeth generally to disregard 
microscopical defects as predisposing causes. Moreover the 
normal fissures of molar and bicuspid teeth are so frequently the 
starting points of caries that it is impossible to ignore the great 
importance of points of least resistance. Dental caries, although 
not a true disease, occurs in a cavity of the body bathed with secre- 
tions physiologically unstable, and containing living cells, all subject 
to various oscillations between disease and health, and we must 
therefore adopt in the study of caries many of the methods applic- 
able to the aetiology of disease in general. We have seen that the 
organisms of diphtheria and pneumonia may exist in normal indi- 
viduals' mouths with no manifestation of disease, that the liberating 
cause of pathological energy, to wit the bacterium, is less than the 
resistance of the body cells, and that it is only in a limited number 
of instances that the balance appears depressed in the favour of the 
bacterium. A little careful consideration will show that dental 
caries has many points of similarity, for in some mouths no caries 
exists, notwithstanding the luxuriant flora present, whilst in others 
apparently similar in all respects caries is rampant. 

Now the teeth themselves cannot be possessed of bactericidal 
action, whereas the fluids of the mouth are daily undergoing physio- 
logical variation, and it seems feasible that alterations in composi- 


tion, reaction or quantity of the buccal fluids may furnish many 
explanatory points in the aetiology of tooth decay. Bacteria are 
notoriously sensitive to their environment, slight changes of medium, 
temperature, alkalinity and what not, favouring the development of 
one species to the exclusion of others ; we may therefore briefly 
conclude that any circumstance or series of circumstances that 
favours the development of acid-forming bacteria and their adhe- 
sion and retention about the teeth, may be rightly considered as 
a predisposing cause of dental caries. 

Food Stuff Chemistry.— Fermentation is of such supreme im- 
portance in dental caries that it will be as well to briefly mention 
some special points relating to fermentative changes. 

Food stuffs are divisible into proteids, carbohydrates, and fats ; 
of these carbohydrates are of the chief importance in caries. 

Carbohydrates are classed in three main groups according to 
their chemical composition : — 

(1) Monosaccharides. 

(2) Disaccharides. 

(3) Polysaccharides. 

Many other carbohydrates exist, but they are physiologically 

(1) Monosaccharides (C 6 H 1 .,0 ). — Dextrose, levulose (glucose). 

These carbohydrates are commonly found in nature, generally 
together, in fruits, seeds, roots and honey. Galactose, another 
carbohydrate of the group, is formed from the hydrolysis of lactose 
or milk sugar. 

They are directly fermentable by yeast into alcohol and carbonic 

C G H 12 O t; + yeast + 2 C,H, . OH + 2 C0 2> 
Dextrose. Alcohol. 

or by several of the schizomycetes of the mouth into lactic acid. 
C 6 H la 6 - -2 CH, . CH . OH . COOH. 
Dextrose. Lactic acid. 

This equation does not exactly express the entire change, as a 
certain amount of the sugar is used up by the growth of the 
organisms. To obtain the acid in pure form a fermentation is 
carried on in a large flask containing sugar (lactose or dextrose) with 
a layer of precipitated chalk at the bottom. As the acid is produced 
it combines with the calcium, forming calcium lactate. The lactate 
is filtered ofl* when the action has ceased, and the acid recovered by 
distillation with sulphuric acid. 


Disaccharides (0 12 H 22 O 11 ). — Cane sugar, milk sugar (lactose), 
malt sugai^" (maltose). 

The disaccharides are regarded as condensation products of the 
monosaccharides with, the elimination of a molecule of water. 

C G H 12 6 + 6 H 12 O 6 = 12 H 22 X1 + H 2 0. 

Dextrose and Lsevulose. Cane sugar. 

The importance of this is seen in the fact that before fermenta- 
tion of the higher sugars occurs they require hydrolising to the 
lower or monosaccharide form. 

Cane sugar is not directly fermentable by yeast, but an invert 
ferment produced by the yeast changes the cane sugar to dextrose 
and laevulose, which is then fermentable. Some organisms occurring 
in the mouth are able to transform the sugar direct, but as a rule 
cane sugar takes much longer to ferment than the glucoses (dextrose 
and laevulose). 

G 12 H 22 O xl + K 2 = 6 H 12 O 6 + C 6 H 12 6 , 
OxaHaaO^ + H 2 - 4 (CH 3 . CH . OH . COOH). 

In the experiment, directly inversion of the cane sugar occurs, 
the solution which before produced no reduction of Fehling's solution 
now gives a marked reaction. 

Maltose ferments readily with yeast and with the majority of 
mouth bacteria. It forms a typical osazone with phenylhydrazine. 
It is the chief sugar formed by the action of ptyalin upon starch in 
the mouth. 

Lactose occurs only in milk. It is the most resistant sugar to 
the effects of yeast, but is fermentable by mouth bacteria, with the 
formation of lactic acid. 

The genus B. lactis, first described by Lord Lister, is composed 
of a large number of different species. Organisms belonging to this 
class are invariably present in milk, and may be generally obtained 
from sour milk by cultural methods. 

Polysaccharides (C 6 H 10 5 )n. — A large group of naturally 
occurring carbohydrates, the chief groups being the starch group, the 
cellulose, the gum group (dextrines, plant gums and mucilages). 

Starch. — Not directly fermentable by yeast ; is fermented by a 
few bacteria occurring in the mouth and intestine. Starch is also 
inverted to maltose by the action of the ptyalin of saliva, and by 
ferments produced and contained in certain bacteria, some of which 
are found in the mouth. 


The chemical changes involved are extremely complicated, but 

the final conversion into maltose may be represented thus : — 

2 C 6 H lo 0, + H.,0 = Cj.Hj.O^; 
Starch. Maltose. 

che fermentation to lactic acid occurring as given above. 
G ia BL 99 11 + H. 2 = 4 CH.CH . OH . COOH 

Under certain circumstances butyric acid may be formed, but 
the quantity is very small. It is generally produced by anaerobic 
bacteria, and may be formed by a direct change of lactic acid : — 
2 C,H 6 3 - C 4 H 8 0, + 2 CO, + 2 H,. 

The other sub-groups of the polysaccharides are unimportant. 

Proteids. — The proteids undergo fermentation by the action of 
bacteria with the production of certain alkaloidal substances, which 
in the presence of air undergo further decomposition. In the 
absence of free oxygen the later changes do not occur, and the 
poisonous alkaloids may become absorbed (Hueppe). 

The digestive effects of bacteria upon proteids are similar to that 
of the pancreatic ferment, and traces of organic acids (paraoxphenyl 
propionic, Sec.) may be formed. They have little importance in 
dental caries. 

Fats. — A certain amount of digestion of fat is produced by 
bacteria with the formation of fatty acids. 

The products are unimportant in dental caries as far as it is 
at present known, and at any rate the amount produced by 
fermentation of fat in the mouth must be exceedingly small in 

The caries of enamel and the caries of dentine present certain 
fundamental differences in their pathology coincident upon their 
different structure, and the mere traces of organic matter contained 
in the former preclude many of the phenomena which may 1)9 
observed in the latter. 

We have already seen that caries is allied in many respects to 
putrefaction, and may in fact be considered as a special case, and 
that the organisms concerned in such a disintegration of tooth tissue 
are by no means of one species and do not necessarily exist in pure 
culture in the decomposing tissues. We have also seen that all 
organisms do not thrive in saliva, and that food stuffs may greatly 
influence the flora at any time present in the oral cavity ; it follows 
then that a large number of bacteria of varying species are to be 
found in decaying dentine, but certain organisms occur more fre- 



quently than others, and we are therefore justified in adopting a 
general grouping of the forms more commonly met with. Some of 
these are members of well-known species, others have so far only 
been described as occurring in the mouth. 

The bacteria are more numerous upon the surface and super- 
ficial layers of decaying dentine than in the deeper layers, while 
enamel holds an intermediate position. Miller first pointed out, 
and I have since confirmed his observations experimentally, that the 
bacteria of tooth decay produce their effects in two ways ; firstly, 
by the production of acids which attack the lime salts of the tooth, 
and secondly, by the development of proteolytic enzymes which 

Fig. 47. — Dental Caries affecting Dentine under Enamel Pit; the minute 


Photomicrograph and specimen by Dr. Leon Williams, x 250. 

digest the matrix denuded of its lime salt, and as a general rale the 
superficial layers contain a preponderance of the liquefying species, 
the deep layers mostly acid-forming bacteria. We are therefore 
able to divide the bacteria of dental caries into two main classes 
corresponding to the predominant biological function : (a) acid 
production ; (b) liquefaction. 

In enamel decay the liquefying organisms are unable to function 


unless they are at the same time producers of acid ; in caries of 
cement both liquefaction and acid destruction may take place 

Caries of Dentine. — The bacteria concerned in the process of 
caries in dentine appear to be greatly influenced by their surround- 
ings, inasmuch as those isolated from the deep layers are generally 
capable of growing under anaerobic conditions — in fact grow better in 
the absence of free oxygen — whereas the surface and superficial layers 
are inhabited by organisms that prefer free oxygen. In making 
cultivations from the deep layers considerable care must be exercised 
to exclude contamination from the surface. The external surface of 
a freshly extracted carious tooth should be well seared with a hot 
iron and the superficial portion of decayed dentine cut away with a 
sterile excavator or knife ; the surface is seared a second time and 
a second slice of dentine removed with a sterile knife. The lower 
layers can now be removed with another sterile instrument, ground 
up in nutrient broth, and cultivations and coverslip preparations 
made from the emulsion. The organisms obtained in cultures by 
this method all appear to be rapid acid producers in the presence of 
carbohydrate food. Arkovy has recently suggested that caries may 
occur in an alkaline medium, and suggests the introduction of a 
third class of " alkali'producers." I do not think there is much to 
commend the adoption of such a division as it appears highly 
improbable that any direct liquefaction of hard undecalcified dentine 
ever occurs through the agency of proteolytic enzymes of bacterial 
origin. Arkovy's ! theory is based on extremely slender evidence, 
without controls and without any proof that acid reaction was 
or was not present in the early stages of the process. In all 
the experiments I have made no liquefaction of hard undecalcified 
dentine was accomplished, although decalcified dentine was digested 
by many bacteria obtained from carious dentine. That liquefied 
gelatin is invariably strongly alkaline is a common fact of laboratory 
knowledge, most organisms will not liquefy acid gelatin in contra- 
distinction to the liquefaction or digestion brought about by most 
ferments of animal origin, which require an acid medium. The 
alkaline reaction of cultures is generally due to ammonia, which 
has no appreciable action upon the tooth salt. The alkali-producers 
of Arkovy are really liquefying organisms, and there is certainly no 

1 Vierteljahrschrift fur Zahnheilkunde, xiv., Heft 3. 


occasion to schedule them twice over on account of their alkaline 

Arkovy's experiment was as follows : two teeth were accidentally 
left in a culture of B. gangraenae pulpse (B. mesentericus var. niger) 
and in some three months came to light by accident. They were 
both carious, and the reaction of the medium was alkaline. From 
this Arkovy deduced primary caries occurring in an alkaline medium. 

In the superficial layers of carious dentine a large variety of 
species are constantly met with, some of them producing diges- 
tive enzymes, others producing acid fermentation, whilst some are 
capable of both functions. Among these liquefying bacteria some 
will dissolve fibrin and blood serum, others only gelatin. The 
ones dissolving blood serum I have found also capable of digesting 
decalcified dentine, while many of those only liquefying gelatin do 
not attack dentine (decalcified). Hard, undecalcified dentine I have 
never found attacked by the enzymes or bacteria. To determine 
the liquefying power of bacteria upon dentine thin strips of decalci- 
fied tooth are suspended in broth cultures of the organism to be 
tested. The strips of dentine should be well washed in dilute 
sodium carbonate and in distilled water after the lime salt has been 
removed by acids. To determine the presence of an enzyme a seven- 
days-old broth culture is poured into a tube containing decalcified 
dentine, and a crystal of thymol or a few drops of chloroform added 
to prevent further growth of the organisms ; a control tube is also 
made containing sterile broth and thymol, with a strip of softened 
dentine. When an enzyme is present the dentine gradually 
dissolves but is unaffected in the control tube. To entirely elimi- 
nate the presence of organisms the culture may be filtered through 
a Pasteur-Chamberland filter and the filtrate containing the enzyme 
tested as before. 

Many of these enzymes may be obtained fairly pure from a 
broth culture by precipitating them with two volumes of absolute 
alcohol filtering, and dissolving up the residue in thymol water. 
I have obtained on several occasions an active enzyme by this pro- 
cedure capable of digesting dentine. The liquefaction of gelatin 
may be conveniently tested by making gelatin tubes containing 
10 per cent, gelatin in saturated thymol water ; the tubes do not 
require cotton wool plugs but are kept mouth downwards in water 
until wanted for use. 

The culture to be tested is poured into one of these tubes, 


and a crystal of thymol added to prevent further growth of the 
organisms ; a control tube is also made containing thymol water 
alone. The tube containing the enzyme shows marked liquefaction 
in a few days. 

I have not found liquefying organisms in the deep layers, with 
the exception of the staphylococcus albus, which organism is 
curiously influenced by its environment, so much so that the 
amount of liquefaction produced by one of the species isolated from 
the deep layers is so slow when first tested that it often requires 
a week or ten days to produce any definite liquefaction. Galloway 
and Eyre ' have shown that by keeping a normal liquefying staphylo- 
coccus albus for long periods under strictly anaerobic conditions, 
the rate of liquefaction is reduced to less than a third of that of 
the control culture grown iierobically. The non-liquefaction of 
organisms isolated from the deep layers of carious dentine is no 
doubt explainable in this way. 

So far I have not obtained anaerobic (obligatory) liquefying 
organisms from dental caries, all the liquefiers being facultative 

Choquet 2 has isolated five different organisms from the recurrent 
caries occurring underneath fillings of three teeth. Unfortunately 
these organisms are not described on the usual test media in use, 
and it is therefore impossible to determine if they are known 
species or not. Choquet does not appear to have found any of 
the organisms observed by other writers, but as he has adopted 
the use of special media only, such a fact is not altogether 

Notwithstanding the profound modification that environment 
and food-stuffs may have upon the flora of the mouth, the organisms 
present in caries are fairly constant ; and adopting the criteria of 
(a) acid production and (b) liquefaction as a rough division of the 
organisms concerned, we may arrange the bacteria of dental caries 
as follows : — 

1 Internat. Med. Congress, Paris, 1900. 
-Dental Cosmos, October, 1900. 



Bacteria of Dental Caries. 

Acid-forming Bacteria. 

Streptococcus brevis 
B. necrodentalis 
Staphylococcus albus 
Streptococcus brevis 
Sarcina lutea 
Sarcina aurantiaca 
Sarcina alba (Eisenberg) 
Staphylococcus albus 
Staphylococcus aureus 

Deep layers of carious dentine. 

Superficial layers of carious tine. 

Superficial layers of carious dentine. 

Bacteria which liquefy Dentine [decalcified). 

None isolated as yet ... Deep layers of carious dentine. 

B. mesentericus ruber 

B. mesentericus vulgatus 

B. mesentericus fuscus 

B. furvus 

B. gingivae pyogenes 

B. liquefasciens fluorescens 

B. subtilis 
Proteus Zenkeri 
B. plexiformis 

Dobrzyniecki, 1 working in Arkovy's laboratory, gives the follow- 
ing list of organisms occurring in carious dentine which agrees 
closely with my own. B. gangraense pulpae (Arkovy) is apparently 
a variety of B. mesentericus, probably "niger." Siberth and myself 
have both come to this conclusion independently, and we have both 
apparently failed to find the organisms described by Arkovy, but 
have constantly met with bacilli of the mesentericus group (potato 

Chief Bacteria of Dental Caries. 


B. gangrsena? pulpse. 
Staphylococcus aureus. 
Streptococcus pyogenes (S. brevis?). 
Sarcinee lutea. 
Staphylococcus albus. 

1 Cent, fur Bakteriol, Bd. xxiii., 1899. 



Pig. 48. Streptococcus brevis. 

Agar cultivation at twentv-four hours. Stained Gram. x 

Fig. 49. — Streptococcus brevis. 
Twenty-four hours' broth cultivation. Stained Gram, 
bourn and Goadby, Odonto. Soc. Trans., 1896.) 

1,000. (Wash- 


I have only found the staphylococcus aureus occasionally, and 
generally the non-liquefying variety. 

A considerable number of chromogenic bacteria are found asso- 
ciated from time to time in dental caries. 

(17) STREPTOCOCCUS BKEVIS (Van Lingelsheim). 
Micrococcus nexifer (Miller). 

Found in the mouth of normal persons as a constant inhabitant, 
generally around the epithelial cells as diplococci, rarely, if ever, 
in chains. Fig. 35. 

Staining Reactions. — Stains by Gram's method and by the 
ordinary aniline dyes. The cocci are generally pear-shaped, and 
rarely show involution forms except in very old cultures. 

Biological Characters. — An aerobic, facultative anaerobic, non- 
motile streptococcus. No pigment formed. 

Gelatin Plates, 22° C. — In forty-eight hours minute grey-white 
flat colonies are formed, which increase slowly and never attain 
a large size. Microscopically : very faintly granular, deep colonies, 
lenticular or morula-like. 

Gelatin Stab, 22° C. — Scanty flat growth upon surface and 
granular beaded growth along the needle track. Occasionally 
liquefaction said to occur. 

Gelatin Shake, 22° C. — Three days : minute colonies scattered 
throughout medium. 

Gelatin Streak, 22° C. — Fairly well-defined grey growth in forty- 
eight hours, with regular edge and minutely granular surface ; 
colonies discrete. According to Lingelsheim, slight liquefaction 
of gelatin takes place, but I have not been able to confirm this. 

Agar Streak, 37*5° C. — Well-marked growth in twenty-four 
hours of minute, moist, grey colonies with no marked centre 
and rapidly coalescing. 

Potato, 37*5° C. — Forty-eight hours : a shining patch of growth is 
seen which may become well marked. No pigmentation occurs. 

Litmus Milk, 37-5° C. — Twenty-four hours : solid clot with well 
marked acid reaction. The greater part of the clot is decolourised 
with the exception of the upper portions. The clot does not 
become re-dissolved. 

Broth, 37*5° C. — Well marked general turbidity in twelve hours ; 
no flocculi are found in the fluid, but a slight granular deposit is 
formed. No indol is produced. No H 2 S. 


Blood Serum, 37 - 5° C. — Similar to agar. 

Broth containing glucose, maltose, lactose, dextrin, inulin, &c, 
shows a marked acid reaction in twenty-four hours. The acidity 
persists for an indefinite time. 

Pathogenesis. — Not pathogenic for guinea-pigs and rabbits when 
injected subcataneously or intraperitoneal^. 


Found in similar situations to the last species. Very common 
m the air. Often present in the mouth and throat. 

Morphology. — Cocci 1 to 1*5 p in diameter associated in pairs 
of hemispherical form, or in packets of four or eight. Sarcina 
form, generally well marked. 

Biological Characters. — An aerobic, liquefying, chromogenic 

Staining Reactions. — Stains by the ordinary aniline dyes and by 
Gram's method. No endogenous spores are formed but arthro- 
spores have been described (Hueppe). 

Gelatin Plates, 22° C. — In two to three days spherical yellow 
colonies appear with projecting central portion, moist, entire and 
opaque. Under the § obj. the colonies are coarsely granular and 
with irregular edge. The colonies grow slowly and do not com- 
mence to liquefy for four or five days. 

Gelatin Stab, 22° C. — A coarsely granular growth of isolated 
colonies (filiform and beaded) along the stab, the colour turning 
a deep yellow. The surface generally shows a distinct button of 
growth before liquefaction commences. The liquefaction occurs first 
as a funnel-shaped depression and gradually spreads to the tube 
w r alls, flocculi of a yellow colour fall to the bottom of the funnel 
and form a granular deposit. 

Some varieties do not liquefy (Chester). 

Gelatin Streak, 22° C. — A well marked layer is formed which 
does not liquefy the medium for several days. 

Agar Streak.— A well marked canary-yellow moist opaque 
growth is formed in three days at 22° C. At 37*5° C. the colour is 
not so pronounced, and the growth tends to a dirty yellow-white, 
and is moist and slightly viscous. 

Broth, 37'5° C. — Little turbidity with yellow deposit in twenty- 
four hours. Indol slight ; a little H 2 S. 

Litmus Milk. — Coagulation of the casein occurs in two days, the 
clot being slowly re-dissolved ; the reaction is strongly acid. 


Potato. — Raised, glistening and limited to streak, surface rough 
and sulphur-yellow in colour. 

Glucose Broth. — Slight acid production. 


Found in air and common in carious teeth, and in gingival 

Morphology. — Cocci 1 to 1-5 /* in diameter, varies considerably, 
occurring typically in packets of eight, but also occurs in pairs of 
large hemispherical elements aud in groups of four (tetrads). 

The elements are smaller than those of S. lutea. 

Staining Reactions. — Stains with the ordinary aniline dyes and 
by Gram's method. Sometimes evidences of a capsule may be 

Biological Characters. — An aerobic facultative anaerobic sarcina, 
growing in the usual culture media at the room temperature and 
forming an orange pigment. Arthrospores said to be formed. 

Gelatin Plates, 22° C. — In forty-eight hours minute spherical 
granular colonies make their appearance. Under the f obj. the 
deep colonies are seen to be somewhat of morula form and may 
become surrounded with a bubble of liquefied gelatin. The surface 
colonies are often two millimetres in diameter, moist, orange yellow 
in colour, raised and often with a central projection (umbonate). 

Gelatin Stain, 22° C. — In two to three days a beaded orange 
growth occurs along the line of puncture, the separate colonies 
ultimately become confluent. Liquefaction commences at the 
surface and takes the form of a deep funnel ; the apex is occupied 
with a granular orange-brown deposit and a good deal of flocculent 
material appears in the fluid. Eventually a scum forms on the 
surface of the liquefied gelatin. 

Gelatin Streak, 22° C. — Liquefaction occurs in three or four 
days ; before this occurs the growth is orange in colour and granular. 

Agar, 37'5° C. — A golden yellow, moist, shining layer is formed, 
the colour being darker than that produced by the staphylococcus 
aureus, and the growth more pronounced and moist. 

Potato, 22° C. — Development occurs slowly and is chiefly con- 
fined to the line of inoculation, and is of brown-yellow colour. 

Blood Serum, 37*5° C. — A well marked layer of dark brown 
colour is formed ; the blood serum may be liquefied in old cultures. 

Broth, 37*5° C. — In twenty-four hours a well marked general 



turbidity, large rlocculi and a thick granular deposit of a brownish- 
orange colour. Indol slight ; no H 2 S. 

Litmus Milk. — In forty-eight hours a well marked acid reaction 
is present and the casein becomes coagulated and later re-dissolved. 

Glucose Broth. — Acid production slight. 


Found widely distributed in milk, water, alimentary tract 
(Vignal), carious dentine, gangrenous pulps, dento-alveolar abscesses 
and gingival inflammation. 

Fig. 50.— Bacillus Mesbntebicus Vulgatus. 
Twenty-four-hours-old cultivation in agar. x 1,000. 

Morphology. — Bacilli 1-2 to 3 /u. long, 075 /* wide, ends rounded, 
generally associated in pairs and in chains (streptobacilli). Often 
long threads are formed, well marked oval spores found, generally 
at ends of bacilli. 

Staining Reactions. — Stains with carbol-methylene blue and 
usual stains, and by Gram's method. Spores stain with hot carbol- 
fuchsin in the usual way. 

Biological Characters. — An aerobic, facultative anaerobic, lique- 
fying, motile bacillus, forming spores. 


Gelatin Plates, 22° C. — In forty-eight hours the colonies are 
transparent, bluish white, but later become opaque with a white 
centre. Liquefaction progresses rapidly, and in three days the 
superficial colonies, which may have attained a diameter of 1 
centimetre, are floating in the liquefied gelatin. Under the f obj. 
th'ey appear granular and with irregular margins, later the surface 
becomes wrinkled and irregular. 

Gelatin Stab, 22° C. — In forty-eight hours a small funnel-shaped 
area of liquefaction has formed. Liquefaction progresses rapidly, 
and in three days has extended to the tube walls. The funnel- 
shaped depression is still seen in the lower part. Flocculi appear 
in the fluid, and a wrinkled pellicle is formed on the surface. The 
funnel generally remains clear except at the apex, which is filled 
with granular deposit. 

Gelatin Streak, 22° 0. — Liquefaction commences in twenty-four 
hours and rapidly progresses, delicate flocculi being formed in the 

Agar Streak, 37-5° C. — A well marked moist dirty grey streak 
with irregular edges is formed in twenty-four hours ; in forty-eight 
hours the surface becomes covered with a wrinkled layer. The 
medium is not stained. 

Blood Serum, 37'5° C— In twenty-four hours a well marked 
groove of liquefaction is formed, which increases rapidly. The 
growth is moist and viscid. Some darkening of the medium may 

Potato, 22° C. — A thick wrinkled white layer is formed all over 
the surface, and generally extending to the back of the slice. The 
culture is thick and stringy, and may be drawn out into threads with 
the platinum needle. 

Broth, 37-5° C. — In twenty-four hours a slight precipitate is 
formed, the fluid remaining clear ; later a thin wrinkled pellicle is 
formed on the surface of the medium, and the deposit becomes 
slightly viscid. The medium does not become turbid. The 
organism will grow in 1 in 200 of hydrochloric acid (Vignal). 

Litmus Milk, 37*5° C. — In twenty-four hours no change ; in forty- 
eight hours coagulation of the casein takes place, but no acid is 
formed. The casein becomes re-dissolved and floats up to the sur- 
face as a slimy layer, the fluid becoming clear, whilst a good deal 
of unpleasant smell is produced. Keaction alkaline. 



Found in company with the foregoing. 

Morphology. — Bacilli 1*2 to 3 h- long, 0-5 /* wide, slightly more 
slender than B. mesentericus vulgatus, but like that organism 
forms streptobacilli and threads. Forms oval spores which are 
highly resistant to heat and germicides. 

Staining Reactions. — Stains with the usual aniline dyes, and 
by Gram's method. 

Biological Characters. — An aerobic, liquefying, motile, chromo- 
genic bacillus. Forms spores. 

Gelatin Plates, 22° C. — At the end of forty-eight hours the 
superficial and deep colonies are different. The former have a 
spreading edge with fine ramifications passing out into the medium, 
the deep colonies are yellow in colour and spherical in shape. 
When they reach the surface they spread out in a similar fashion to 
the deeper ones. Liquefaction commences about the third or fourth 
day, and the colonies float on the surface of the fluid, the radiat- 
ing process disappearing, and the surface becoming dark and 
wrinkled. The medium becomes stained a light brown. 

Gelatin Stab, 22° C. — In three or four days growth has developed 
to the bottom of the stab, liquefaction commencing as a funnel- 
shaped depression at the surface, and rapidly spreading to the walls 
of the tube (saccate). The funnel remains below the liquefied 
gelatin, nocculi forming as in the other members of the group. 

Gelatin Streak, 22° C. — In two days a well marked liquefied 
groove is formed. 

Agar Streak, 37-5° C. — A dirty yellow or white crumpled layer 
is formed all over the surface in twenty-four hours, which becomes 
dry in old cultures. 

Blood Serum, 37*5° C. — In two days well marked liquefaction 
has taken place, the medium becoming darkened. 

Potato, 37*5° C. — In twenty-four hours a viscid yellowish-white 
or pink layer has formed covering the whole surface of the slice ; 
in two days the growth has extended round the whole of the potato, 
which is coloured a rose pink. The extension to the back of the 
slice is characteristic. At 22° C. the layer is more yellow, viscid, and 
does not turn pink. 

Broth, 37*5° C. — Flocculent deposit in twenty-four hours, later 
a slight pellicle is formed which has a brown wrinkled surface. 

Litmus Milk, 37° C. — Coagulation of the casein occurs in two 


to three days, the coagulum is re-dissolved, and the fluid clears. 
Grows at any temperature from 15° to 40° C. 


Found in similar situations to the last. 

Morphology. — Bacilli 1*3 to 3*5 m long, 0-5 a* broad, ends rounded; 
forms chains and threads. 

Staining Reactions. — Stains with the ordinary aniline dyes, and 
by Gram's method. Spores stain in the usual way. 

Biological Characters. — An aerobic, liquefying, motile, spore- 
forming bacillus. The spores are irregularly placed in the rods, 
and are rather smaller than those of the other two species. 

Gelatin Plates, 22° C. — In two days spherical white colonies are 
found which have radiating processes extending into the medium. 
The superficial colonies particularly have a fine granular surface. 
Liquefaction rapidly occurs, the medium turning brownish in tint. 

Gelatin Stab, 22° C. — In forty-eight hours a beaded growth 
appears to the bottom of the stab. Liquefaction commences at 
the surface in a funnel shape and rapidly reaches the walls of 
the tube. Flocculi form in the medium, and a pellicle on the 

Gelatin Streak, 22° C. — A liquefied groove appears in two to 
three days filled with flocculi. 

Blood Serum, 37*5° C. — A groove of liquefaction with discoloura- 
tion of the medium occurs in two days. 

Potato, 37° C. — A well-marked rugose yellowish-brown layer is 
formed of wash-leather colour ; the potato becomes stained. The 
growth is thin and does not penetrate so much as does B. mesen- 
tericus ruber or vulgatus. A good deal of sour putrefactive odour is 
given off from the potato and blood serum cultures. 

Broth. — 37*5° C. — A flocculent precipitate is formed, and later 
a surface pellicle, brown and wrinkled. 

Litmus Milk, 37'5° C. — Coagulation of casein occurs, the 
coagulum eventually becoming re-dissolved and floating on the sur- 
face of the fluid, which clears. The reaction is alkaline, the fluid 
turning a bluish-brown. Some gas is formed. 

(Bacterium termo of Vignal. ) 
Found in water and in various putrefying infusions ; often present 
in the mouth and sputum. 


Morphology. — Bacilli 1*5 to 2 h- long, and 0*3 /* broad ; often 
united in pairs with a central constriction ; grows out into filaments. 

Staining Reactions. — Stains with the ordinary aniline dyes, but 
not by Gram's method. 

Biological Characters. — An aerobic, facultative anaerobic, chromo- 
genic, liquefying, motile bacillus ; does not form spores. 

Gelatin Plates, 22 G C. — In forty-eight hours white colonies are 
formed which may attain a diameter of 2 mm., a ring of liquefied 
gelatin forming around each ; under f obj. the colonies have a well- 
defined, sharp outline with a circular dentate edge. The centre 
of the colony is darker and brown in colour, finely granular ; the 
outer zone is paler and yellow in colour, finely granular, and 
becoming translucent towards the edge. The surrounding gelatin 
gradually becomes a fluorescent green by transmitted, yellow by 
reflected light. 

Gelatin Stab, 22° C. — A white granular growth quickly appears 
along the line of puncture, a small funnel of liquefied gelatin 
appearing at the surface, the widest part of which is usually 
occupied by an air bubble. The liquefaction gradually extends to 
the sides of the tube and at the same time in a downward direction, 
the liquefaction ultimately taking place in a horizontal plane 
(stratiform). Just below the area of liquefaction, and spreading 
into the surrounding medium, is a delicate fluorescent green 
colouration which gradually spreads throughout the tube. The 
colour is green by transmitted, yellowish-brown by reflected light. 
A thick white deposit is formed at the bottom of the liquefied 

Agar, 37*5° C. — A slimy, moist, grey-white layer is rapidly formed ; 
at 22° C. the fluorescent green pigment is produced as on gelatin and 
colours the whole of the medium, which later becomes of a dirty 
yellow-brown. The growth itself is not coloured. 

Potato, 22° C. — A well marked brownish layer is developed and 
the potato stained a dark tint. The fluorescent green colour may 
be extracted with chloroform from the potato cultures. 

Blood Serum, 37"5° C. — Three days : dark and discoloured groove 
of liquefaction. 

Broth. — 37*5° C. — Twenty-four hours : general turbidity with 
thick precipitate. Indol is formed in small quantities, but no H 2 S. 

Litmns Milk, 22° C. — Not coagulated, but clears up gradually 
with yellowish-green colouration. 



Bacillus fluorescens non liquefasciens is also often found in the 
mouth. As its name implies, it does not produce liquefaction of 
gelatin or blood serum, and is non-motile. The green fluorescent 
pigment produced is often seen in broth cultivations, especially so 
when the culture is shaken up with air, the colour disappearing 
again on standing. 

The other biological reactions are similar to the organism 
described above. Euzicka, 1 after a prolonged examination of this 
group of bacteria and the B. pyocyaneus, came to the conclusion 
that they were all varieties of one species, although perhaps modified 
by their surroundings, &c. Lehmann and Neumann 2 also support 
this view. There are a considerable number of organisms described 
as producing a greenish fluorescence, and it is certainly not im- 
probable that they are also members of this one species. 

Fig. 51.— Bacillus Subtilis, showing Spoee Formation, x 1,000. 

(24) BACILLUS SUBTILIS. (The Hay Bacillus.) 
Found in soil and water, very common. Often associated with 
the Mesenteric group in the mouth and in carious dentine. 

Morphology. — Bacilli 4-6 to 6 ^ long, 0*75 to 1 v- broad, with 
rounded ends. Often associated in chains (streptobacilli). Some- 
times grows out into very long filaments, especially in liquid media. 
Flagella multiple (peritrichic). 

Arch, fur Hygiene, Bd. xxxiv., p. 148. 
Bacteriology, p. 286. 


Staining Reactions. — Stains with the ordinary aniline dyes, and 
by Gram's method. The spores stain well by Moller's method. 
The flagella are stained by Pitfield's method. 

Biological Characters. — An aerobic, liquefying, motile bacillus. 
Forms spores, which are generally situated at the ends of the rods. 
The spore germination is characteristic. The germinating spore 
splits along one side, and the organism grows out through the rent, 
the remains of the spore often remaining attached to the end of the 
bacillus. The motility is of a curious waddling nature, and is not 
very rapid even when the bacillus possesses many flagella. 

Gelatin Plates, 22° C. — In twenty-four hours minute grey-white 
colonies appear under the | obj., they are granular, greenish, and 
have a well defined but irregular outline. Development progresses 
rapidly, and in two days well marked liquefaction has taken place, 
forming saucer-like cavities with granular, translucent contents ; 
the central part being opaque and white. Under the § obj. the 
colonies are greyish-yellow in the centre, and greenish-grey towards 
the periphery where a tangled mass of filaments is to be seen, which 
radiate into the surrounding medium and also into the non- liquefied 
portion (crateriform, turbid, entire, becoming ciliate). 

Gelatin Stab, 22° C. — A white growth rapidly appears along the 
needle track (saccate), and liquefaction soon commences with the 
formation of a wrinkled mycoderma upon the surface. The pellicle 
thus formed sinks to the bottom of the liquefied gelatin, and is 
replaced by another which in turn sinks, so that a thick deposit is 
formed at the bottom of the tube. The fluid which at first is filled 
with white flocculi becomes clear as the result. Occasionally the 
liquefaction does not progress so rapidly, and fine radiating processes 
extend into the non-liquefied medium, which disappear as liquefac- 
tion progresses. Various races of subtilis show considerable variation 
in their liquefying power. No gas is formed. 

Agar Streak, 37"5° C. — In twenty-four hours a grey, opaque flat 
growth with defined edges, which later becomes dry, irregular and 
brownish in colour. The whole may be often lifted away from the 
surface of the nutrient medium. The surface, at first slightly 
mottled, becomes corrugated and wrinkled (crumpled). 

Potato, 37-5° C. — In twenty-four hours the whole surface of the 
slice is covered with a moist creamy growth which extends on to 
the glass of the culture tube. The growth is found to be full of 
spores when examined microscopically. 


Blood Seru?n, 37*5° C. — A wrinkled mycoderma is rapidly formed 
over the surface, and liquefaction of the medium occurs. 

Broth, 37*5° C. — In liquid media general turbidity is rapidly 
formed, and the characteristic wrinkled mycoderma is formed upon 
the surface, and becomes firmly attached to the tube walls. On 
vegetable infusions of all kinds a similar wrinkled pellicle is rapidly 
formed, and is seen to consist of tangled threads when examined 
under the microscope. Indol negative. 

Litmus Milk. — An acid reaction is generally produced, and the 
casein is dissolved. Later the reaction becomes alkaline. Various 
sugars are oxidised, saccharose inverted and then oxidised. The 
process is continuous if the acid formed is neutralized as formed 
with precipitated chalk (Lefar). 

The organism is extremely sensitive to acids, and in the presence 
of minute traces forms all sorts of curious involution forms (see 
fig. 3). 


The common bacterium present in putrefactive processes ; widely 
distributed. This and other putrefactive bacteria were once de- 
scribed under the term " Bacterium Termo." 

Morphology.- — Bacilli with rounded ends, 0-8 fi broad, of variable 
length (1*5 to 3 h) ; long filaments 20 as and longer are formed ; 
these filaments may be flexible and sometimes spiral in form. The 
rods are frequently united in pairs ; they are motile and have 
peritrichic flagella (fig. 3 (iii.) a). Involution forms are common, the 
most frequent being of globular shape, and are generally found 
in cultures incubated at 37*5° C. Spore formation not observed. 

Staining Beactions. — Stains well with the ordinary aniline dyes, 
but does not retain the stain of Gram's method. 

Biological Characters. — An aerobic, facultative anaerobic, liquefy- 
ing, motile bacillus growing in the usual culture media at the room 
temperature. Optimum temperature 22° C. 

Gelatin Plates, 22° C. — In 5 per cent, gelatin in eight hours 
small depressions are seen on the surface of the medium. Under 
the microscope these depressions are seen to have amoeboid-like 
processes extending on to the surrounding surface. These processes 
undergo constant change in form, and may become detached and 
wander over the plate (Sternberg). 

The deep colonies have radiating processes extending into the 


unliquefied gelatin. No wandering colonies are formed in 10 per 
cent, gelatin. 

Gelatin Stab, 22° C. — Liquefaction takes place to the bottom of 
the puncture and rapidly spreads to the walls of the tube (saccate) ; 
near the surface a white cloudiness is produced and an abundant 
flocculent deposit is formed at the bottom. 

Gelatin Shake, 22° C. — Abundant gas formation and liquefaction 
of gelatin. 

Blood Serum, 37*5° C. — Dirty white growth in twenty-four 
hours with well-marked liquefaction of medium, which becomes 

Agar, 37'5°C. — In twenty-four hours a spreading, pale yellowish, 
glistening, translucent growth covering the whole surface of the 

Potato, 37 ; 5° C. — In twenty-four hours a dirty white moist growth. 
The surface of the potato becomes dissolved. Putrefactive smell 
and alkaline reaction. 

Litmus Milk, 37*5° C. — Slightly acid in twenty-four hours, later 
distinctly alkaline, but no clotting takes place. The casein becomes 
dissolved, and the fluid ultimately is clear with a thick precipitate 
of a brown-blue or yellow colour. 

Broth, 37*5° C. — General turbidity and precipitate in twenty-four 
hours ; there is generally a putrefactive smell. Indol formed. H 2 S 

Nitrate Broth. — Well marked reaction with naphthylamine (see, 
page 226). 

Glucose Broth.— Gas formed c " ■ f ', no gas in lactose broth. 

Pathogenesis. — Pathogenic for" rabbits and guinea-pigs when 
injected into the veins in considerable quantities. Cheyne estimated 
that a cubic cm. of liquefied gelatin contained 4,500,000,000 bacilli, 
and that a smaller dose than 9,000,000 produced no ill effect. The 
organism has often been isolated post mortem from venous thrombi. 1 
Filtered cultures cause toxaemia. 

Found in dental caries, especially in the deep layers of carious 
dentine, where the rods may be found blocking up the dentinal 
canal. When first isolated it grows best in an atmosphere free from 

1 Bryant, Tram, Path. Soc, 1901. 



oxygen, but is facultative aerobic, and grows well under ordinary 
conditions. It develops well on the ordinary laboratory media used. 
Morphology . — Bacilli 0*75 fx broad, and 1 to 5 /* long, often asso- 
ciated in pairs and sometimes in chains (streptobacilli). The ends 
of the bacilli are square or rounded. In anaerobic cultures the 
bacilli tend to grow out into long threads ; under aerobic conditions 
the organism is much shorter. The bacilli tend to involute rapidly, 
and form swollen and contorted masses not unlike the streptococcus. 

Fig. 52. — Bacillus neceodbntalis. 
Agar cultivation, forty-eight hours. Stained Gram, x 1,000. 

In broth cultures the bacilli are very short and appear more 
like cocci. They are slightly motile, best marked on anaerobic 
cultures. I have not succeeded in staining the nagella. 

Staining Beactions. — Stains by the ordinary aniline dyes but 
takes some time, especially with methylene blue. It retains the 
stain of Gram's method. The involution forms stain badly and 
appear granular, but no polar staining has been observed. 

Biological Characters. — An anaerobic, facultative aerobic, non- 
liquefying motile bacillus. No spore formation observed. Non- 


Gelatin Plates, 22° C. — In three days minute colonies appear, 
not much larger than a pin's point. The colonies spread a little, 
aDd feathery processes extend into the medium. The deep colonies 
are often surrounded with a series of fine rays. No liquefaction of 
the gelatin occurs. 

Gelatin Streak, 22° C. — In three days a slight beaded growth 
occurs, which later sends processes into the medium. The gelatin 
is not liquefied, but may become a little softened around the 

Gelatin Stab, 22° C. — Slight granular growth along the line 
of puncture, radiating processes may be formed. No liquefaction 

Gelatin Shake, 22° C. — No gas bubbles are formed. 

Potato, 37*5° C. — In forty-eight hours slight shining appearance 
upon the inoculated surface. The organisms show considerable 

Agar Plates, 37*5° C. — Minute grey colonies in forty-eight hours, 
round and regular or erose edge, and central nucleus brownish 
and raised. Microscopically, brownish-yellow with central nucleus 
faintly granular and regular edge. Under anaerobic conditions the 
colonies are larger. 

Broth, 375° C. — In twenty-four hours slight general turbidity 
with a flocculent precipitate, which increases whilst the turbidity 
does not. No pellicle is formed. No indol produced in ten days ; 
no H 2 S formed. 

Litmus Milky 375° C. — In twenty-four hours no change ; in 
forty-eight hours solid clot, lower portion decolourised, the top 
showing a marked acid reaction. The clot is not re-dissolved. 

Glucose broth, lactose broth, starch broth, maltose broth : 
strong acid reaction in forty-eight hours. 

Aivierobiosis. — Well marked growth on glucose formate agar in 
Buchner tubes. The colonies are much larger than on aerobic 
media, and are brownish in colour and have a well marked nipple- 
like central projection (umbonate). No gas is produced on glucose 
formate broth, but the turbidity is well marked. 

Pathogenesis. — Not determined. 

Spore Formation. — No spores stainable. Cultures three weeks 
old, heated to 70° C. for half an hour, gave no subsequent growth. 

Optimum temperature, 37*5° C. Thermal death point 60° C. 



Fig. 53. — Bacillus plexiformis (Goadby). 
Gelatin cultivation at forty-eight hours, x 1,000. 

Fig. 54. — Bacillus plexiformis. 
Twenty-four hours' cultivation of decalcified dentine, x 1,000. 



Found occasionally in carious dentine. 

Morphology. — Curved and twisted bacilli on most media ; may be 
associated in pairs or grow out into long threads 30 fi or more 
long. In gelatine cultures the bacilli are short and tend to stain 
irregularly (see fig. 53), while on slices of decalcified dentine long 
threads are formed (see fig. 54). Motility well marked but flagella 
not stained. 

Staining Reactions. — Does not stain by Gram's method, stains 
by ordinary aniline dyes. No spores observed. 

Biological Characters. Gelatin Plates, 22° C. — Minute white to 
grey colonies (punctate or effused). Gradual liquefaction occurs. 

Gelatin Stab, 22° C. — Filiform growth to bottom of stab, lique- 
faction only in upper part. Stratiform, well marked flocculent 
deposit and general turbidity of fluid. 

Gelatin Streak, 22° C. — Well marked liquefied groove in two to 
three days. 

Agar Plates, 37*5° C., 2 to 3 mm. — Translucent colonies raised 
and round (pulvinate), edge entire, of rather viscous consistency. 

Agar Streak, 37*5° C. — Eaised, moist, gummy, confined to needle 

Blood ' Serum, 37*5° C. — Dirty brown streak, eventually slight 

Potato, 22° C. — Brownish slimy growth, slow in appearance, 
confined to streak. Not spreading. 

Litmus 21 ilk, 37*5° C. — Slightly alkaline reaction, no clot. 

Broth, 37*5 C°. — General turbidity with somewhat flocculent 
deposit, no pellicle. Indol reaction well marked. 

Glucose formate media. — Xo anaerobic growth and no gas bubbles 


Bacteria in Tooth Pulps. 

The channel of infection of the tooth pulp is along the dentinal 
canals, and may occur with but slight and almost imperceptible 
signs of caries in the tooth surface (see fig. 47, inroads of organisms 
well shown). 

When once the dentine has been reached the organisms are able 
to make their way along the dentinal tubules, at the same time that 
their products penetrate to the pulp surface by capillarity. I have 
often found that cultivations and microscopical examination of pulps 
which had succumbed to caries showed no bacteria, while the den- 
tine at a short distance from the pulp chamber gave positive results. 
Miller has several times pointed out that cultivations made from 
tooth pulps gave no evidence of living organisms even after most 
careful examination. The soluble products of bacterial activity 
may therefore produce death of the pulp, accompanied with fatty 
or other degeneration, without the living organisms themselves 
actually coming into play, and the familiar clinical observation of the 
ease with which arsenious acid gains access to the pulp demon- 
strates the permeability of sound dentine. 

In the largest number of cases, however, bacteria are present in 
dead pulps ; some of them have been found by Miller to be patho- 
genic for animals, generally producing local necrosis or suppuration 
when injected subcutaneously. Miller examined fifty cases of pulp 
gangrene, and notes several of the organisms met with. Many of 
these are gas-forming bacteria, and most of them capable of ferment- 
ing carbohydrates. Among the known pathogenic bacteria that 
have been found in tooth pulps, the streptococcus has been observed 
by most of the workers : Miller, 1 Siebeth, 2 Dobrzyniecki 3 and myself 
have constantly met with it, but I am inclined to think that it is the 

1 Dental Cosmos, July, 1894. 

- Central, filr Bak., 1900, xxviii., p. 302. 

3 Central, fur Bak., 1898, xxiii., p. 670. 


ordinary streptococcus of the mouth (S. brevis) rather than the 
pathogenic streptococcus. 

Miller also met with Micrococcus tetragenous, whilst Zierler 1 
occasionally found sarcinae. 

All these organisms are met with from time to time in carious 
dentine, so that their presence in dead pulps is not surprising, and I 
can confirm the occasional presence of both sarcinae and micrococcus 
tetragenous from my observations. Staphylococci are not infre- 
quent, the most common variety being the Staphylococcus albus, 
although the staphylococcus aureus does also occur. I have occa- 
sionally (four times), met with S. aureus in pure cultivations in the 
abscesses of roots in which the pulp had been dead for a consider- 
able time. B. necrodentalis is also often obtainable, as is B. gingivae 
pyogenes. The cultural characters of these bacteria have been already 
given (pages 127 and 161) in the chapter on Dental Caries. 

Miller, in the paper already referred to, gives the following list 
of four organisms which he has frequently met with : — 

(1) Cocci and diplococci (pathogenic). 

(2) Bacilli, curved and often forming threads. 

(3) Short rods with bipolar staining. 

(4) Micrococcus tenuis. 

He found these organisms frequently present, but does not state 
how often in pure culture, or in combination with the others. 

A number of other organisms were also observed, some of which 
grew upon agar, others upon gelatin, but it is not clear which. The 
cultural reactions were unfortunately omitted. 

Miller concluded that the cocci present were probably more 
concerned in pulp destruction than the bacilli, but that some 
symbiotic relation existed between the bacilli and cocci ; injection 
of animals with mixed cultures producing more marked effect than 
the pure cultures alone. Inoculation with masses of putrid pulps 
generally resulted in the death of the animals (mice and rats) in 
three or four days, with local tissue necrosis and occasionally 

Miller did not meet with the pneumococcus during any of his 
investigations ; although more than one hundred and fifty mice 
were inoculated with putrid pulps, in no case did the pneumococci 
appear in the animals' blood after death. 

Schreier 2 claims to have observed the pneumococcus in dead 

1 Med. Rundschaw Ber., 1900, p. 534. 

2 Oest. Vng. Vietelj. fiir Zalinarzte, 1893, Heft ii. 


pulps ; two experiments only were made, and the inoculations 
performed with two broth cultures ; one animal died, the others 
did not. 

From the experiments of Miller it is extremely improbable that 
the pneumococcus is a constant organism in the diseased dental 
pulp, and from the particulars given by Schreier it is also highly 
improbable that the organisms he isolated w T ere pneumococci. 

Arkovy, Zierler and Sieberth have all noted the presence of 
bacilli of the Mesentericus group (potato-bacilli) in the gangrenous 
pulps. Arkovy first described an organism which he named B. gan- 
grsenae pulpae as constantly present in putrid pulps, the description 
appearing in the Cent, filr Bak., Bd. xxiii., p. 962, 1897, and he there 
described the organism in question as belonging to the Proteus 
group on account of its pleomorphism, in that it formed cocci on one 
medium and bacilli on another. Unfortunately further research 
resulted in the discovery that the supposed cocci were spores. 

The supposed cocci were however originally figured stained by 
methylene blue, although they were said to stain but lightly. As 
the method of spore staining and resistance to high temperatures 
was also given, it is difficult to understand how the mistake arose, 
particularly as spores can be easily observed in hanging-drop 
specimens with a J obj. on account of their highly refractile nature. 
Arkovy 1 in a later paper alters his classification of the organism and 
admits it belongs to the Subtilis group. 

Zierler, a little later than Arkovy, described an organisism which 
Arkovy is at some pains to point out differs from B. gangraenae pulpa? 
in that it does not produce so much colour, and that the liquefaction 
of gelatin is slower, while upon potato the organism described by 
Zierler produces a rose tint, whereas B. gangraenae pulpae turns the 
medium dark brown. 

Otto Sieberth and myself have both noted the constant presence 
of bacilli of the Mesentericus group in pulps and carious dentine, 
and Sieberth is strongly of the opinion that B. gangraenae pulpae is 
one of the same group, and I quite agree, for in the bacterio- 
logical examination of a large number of dead pulps I have 
not met with any bacilli with such frequency as the Mesentericus 
group. Three marked varieties of this group are to be met 
with : (1) B. mesentericus vulgatus ; (2) B. mesentericus ruber ; 

1 Cent, fur Bahteriol., Bd. xxix., No. 19, p. 745. 


(3) B. mesentericus fuscus. The last corresponds in all particulars 
to B. gangraenae pulpae, whilst the second is without doubt the 
organism found by Zierler. 

The spores of these organisms are highly resistant and withstand 
boiling for half an hour or more. 

The biological description of the " Potato bacilli," as they are 
termed, is given as well as that published by Arkovy of B. gangraenae 


Bacteria in Dento-Alveolar Abscesses. 

The bacteria found in the acute abscesses arising from dead 
pulps belong as a rule to the cocci, but it is somewhat remarkable 
that the majority of cases do not give cultivations of the common 
pus cocci. 

Staphylococcus aureus is the least often met with in my 
experience. In twenty cases examined carefully S. aureus occurred 
in three, and on another occasion it was present in pure cultivation 
in the pus of a dead tooth, only a slight local inflammation being 
present, with little or no swelling and no pointing through the 

There appears to be more than one organism concerned in the 
process that may, after gaining access to the alveolus by way of 
the pulp chamber, produce a chronic inflammatory condition of 
the tissues in the region of the apex, remaining more or less 
quiescent until some secondary cause stimulates them into activity. 
One organism in particular, a coccus, I have frequently found in 
pure culture in abscesses involving teeth. The determination of 
the organisms present in such abscesses as have not pointed through 
the alveolus is simple ; the root canal and pulp chamber are 
sterilized with hot instruments and a wisp of sterile wool intro- 
duced, and a cultivation made from the apical portion in one or 
two days' time. The colonies of the coccus are extremely gelatin- 
ous upon agar, and are most difficult to remove except en masse. 
The organism is described below. 

Staphylococcus albus is the next most frequent organism met 
with in dento-alveolar abscess, and occurs in about half the cases, 
whilst Micrococcus tetragenous may also be sometimes found. 

In some abscesses, more particularly those severe forms result- 
ing in pyaemia or cellulitis, streptococci have been found by several 


observers ; ' whilst in a series of twenty cases I examined consecu- 
tively, B. coli was present in two with particularly foetid pus. 
A variety of alveolar abscess occurs occasionally around those teeth 
affected with pyorrhoea alveolaris ; in these cases the coccus I have 
described below is frequently found, as are many of the other 
bacteria* present in the exudate occurring along the gum margins. 
Kirk - made an investigation of the bacteria present in the small 
abscesses occasionally associated with living teeth. He found in 
some cases a diplococcus which had many of the characters of 
the pneumococcus, from which organism however it differed in 
many respects. The matter requires further investigation. 

A variety of dento-alveolar abscess, fortunately not common, 
has a great tendency to spread and infiltrate the surrounding 
tissues. I have investigated four abscesses of this sort, but have 
been unsuccessful in isolating an organism which appears to be 
constantly present. It is anaerobic, and on the glucose formate 
stabs produces a considerable quantity of gas, so much so that the 
agar is often broken up and blown to the top of the tube. The 
microscopical examination shows bacilli forming long threads and 
showing a tendency to stain irregularly with methylene blue. As 
the organism was not isolated it is impossible to make any further 
statements. Staphylococcus albus was the only orgauism obtained 
in pure culture from these four cases. One other series of micro- 
organisms often associated with dental abscesses deserves mention, 
namely, the Blastomycetcs. 

It is not at all uncommon to find yeasts present in the chronic 
abscesses associated with dead roots which have been for a long 
time exposed to infection from the mouth, such roots, for instance, 
as have widely open root canals. So far I have not investigated the 
species, but it is interesting to note that recently a good many 
observers have described pathogenic blastomycetes which are 
capable of curious tumour formation (ef. the one described by Klein 
in the Trans. Pathological Society, 1901). In chronic abscesses 
Arkovy has constantly found the organism B. gangraenae pulpae 
( ? Mesentericus) already discussed. 

Altogether the bacteriology of dento-alveolar abscess is 1)}- no 
means fully worked out, and the literature of the subject is extremely 

1 Roughton, Trans. Odont. Soc, November, 1891. 
- Cosmos, 1901. 



small. In general it would appear that the bacteria associated in 
the process are as a rule cocci, but that any organism or organisms 
able to set up suppurative pulpitis might be a cause of the abscess 
developing later. It also seems highly probable that some organisms 
hitherto undescribed are concerned in some of the more virulent 
forms of alveolar suppuration. 

That many organisms of a pathogenic nature gain access to the 
body through the portal of diseased teeth is admitted by most, and 
that such bacteria produce chronic inflammatory conditions of th< 
cervical glands is also well known, but concise bacteriological know- 
ledge of the process has yet to be gained. 

Fig. 55. — Yeast Forms. 
Showing development of mycelium (from Eyre's " Bacteriological Technique "). 


Common in dento-alveolar abscesses and in suppuration along 
the gums; occurs occasionally in antral suppuration. Often asso- 
ciated with bacilli of Mesenteric group. 

Morphology. — Irregular cocci, 075 to 1 m wide. Sometimes 
arranged in fours, generally as isolated cocci or as staphylococci. 

Staining Reactions. — Stains by ordinary aniline dyes and by 
Gram's method. There is a considerable amount of gelatinous 


material around the organisms, which tends to stain with the ordinary 
dyes and is of a mucinous nature. 

Biological Characters. — An aerobic, non-liquefying coccus ; non- 
mobile. No spores formed. * 

Gelatin. — Does not grow at room temperature ; grows slowly at 
37 c C. 

Agar Plates, 37-5° C. — Flat spreading colonies, dentate edge, 
surface granular ; microscopically, surface colonies, irregular dentate 
edge, brown to brownish-black, surface granular, marmorated 
(veined): Deep colonies, irregular, moruloid, brown. 

Agar Streak, 37 5° C. — Two days : raised viscous shining streak, 
edge defined, wavy. The whole mass may be wound up upon the 
platinum needle and is extremely viscous. 

Potato, 37'5° C — Slight flat grey viscous growth confined to area 
of inoculation. 

Blood Scrum, 37*5° C. — Similar to agar; no liquefaction. 

Litmus Milk, 37'5° C. — No change. 

Broth, 37'5° C. — General turbidity with well marked pellicle and 
flocculi floating in fluid and deposit. 

Peptone Water, 37'5°C. — H 2 S formed. Glucose, dextrin, starch, 
lactose, acid in three days. No nitrite and no indol ; gas evolved 
with nitrate media. 

Glucose formate media, 37. 5° C. — No growth anaerobically, but 
development when air is admitted 


Ulcerative Stomatitis.— Nothing is at present known of this con- 
dition as far as its bacteriology is concerned. It occurs with many 
diseases associated with fever and it appears to spread from the 
gum margins ; particularly is this the case in the mouths of those 
already suffering from marginal inflammation and pyorrhoea alveo- 
laris. Many of the mouth organisms are increased in numbers in 
the condition, especially the spirilla, and I can confirm Bernheim's 1 
observations that these organisms are constantly present in the 
disease. Ulcerative stomatitis also occurs in an epidemic form, 

1 Scmainc Medicate, 1897, p. 252. 


and it has been suggested 1 that it is related to the " foot and mouth 
disease " of cattle. 

Foot and mouth disease has been investigated by Loftier 2 and 
Trosch, who found that the cause was present in the vesicles and 
the mouth secretions. Moreover it required several nitrations 
through porcelain niters to remove the active agent, which so far 
is invisible to the most powerful microscopes. The lymph filtered 
once is still capable of producing the disease when inoculated into 

Aphthous Stomatitis is also without bacteriological investigation. 

Gangrenous Stomatitis. — Petruschy 3 has found diphtheria bacilli 
together with pseudodiphtheria bacilli in two cases, which were 
cured with injection of diphtheria antitoxine. The condition occurs 
as a sequela of various fevers. 

Mycosis of Tonsil and Mouth. — Occasionally large patches of a 
white numular nature are formed upon the tonsil and buccal mucous 
membrane. Sometimes these nodular masses are composed almost 
entirely of sarcinse, at other times they are found to consist of 
tangled masses of threads (Leptothrix ?) and various other bacteria ; 
yeasts are also frequently present, sometimes to the extent of a 
false membrane. I have met with two such cases. 

Epidemic Parotitis. — Laveran 4 and Mercay 5 and Walsh have 
found diplococci resembling staphylococcus albus in cases of mumps ; 
the injection produced transitory orchitis in rabbits and dogs, with 
occasional parotitis. The matter requires confirmation. 

Suppurative Parotitis occurs occasionally associated with 
intestinal growth and gastric ulcer. In one case which came 
under my own observation staphylococcus aureus was present in 
pure culture, in another staphylococcus albus and a bacillus form- 
ing long threads which died out before its biology could be 

1 Osier, " Princ. of Medicine," p. 442. 

2 Cent, fur Bakt., Bd. xxiii., 371. 

3 Deut. med. Wochenschr., 1898, 600. 

Com]), rend. Soc. Biol., 1893. 5 Cent, fiir Bakt., xxi., I 


Pyorrhoea Alveolaris. 

Chronic suppurative periodontitis, caries alveolaris specifica, 
Rigg's disease, periostitis alveolo-dentalis, &c, &c., are among the 
terms applied by various authors to the chronic inflammatory con- 
dition of the gum margins and peridontal membrane leading to 
wasting of the alveolus and loss of the teeth. A coverslip prepara- 
tion made from the pus found in the pockets around the teeth of 
chronic suppurative periodontitis shows a large number and variety 
of morphological forms so varied and changeable that there is 
considerable difficulty in tabulating them. Cocci as a rule are 
present in only small numbers in coverslip specimens, but in the 
usual culture media cocci invariably develop, even when plate 
cultivations are made from the mouth direct the majority of the 
colonies appearing belong to the genus cocci. Anyone who has 
been engaged for any length of time on the study of mouth bacteria 
cannot fail to have been struck with the difficulty of recovering 
in pure culture the organisms seen to be present in the pus of 
pyorrhoea. The staphylococcus viscosus described in the chapter 
on caries is frequently present, and from its constant presence in 
dento-alveolar abscesses may have some share in the pus formation. 
Most of the morphological forms met with develop for a period on 
maltose-agar, but it is well nigh hopeless to obtain pure cultures by 
the ordinary process of plating. 

The organisms seen on direct examination may be tabulated as 
follows : — 

(1) Cocci — generally in diplococci and massed around the 
epithelial cells in clumps. 

(2) Thick bacilli generally jointed and often showing consider- 
able irregularity in their staining characters. 

(3) Thick bacilli with pointed ends and somewhat of the shape 
of a bean pod ; they frequently show a division in the centre and 
appear as two elongated triangles with the bases opposed. 


(4) Various fine bacilli 0-5 p and under in width often exhibiting 
an irregular banded marking, especially well marked in the larger 
threads, which may be of great length. 

(5) Spirilla, spirochete, and comma-shaped bacilli, all showing 
marked motility in the hanging drop. 

(6) Various yeast forms, sometimes with a partially developed 

(7) Streptothrix threads, generally showing well marked clubs 
(see fig. 67). 

(8) Masses of bacilli associated with the threads, some jointed 
in chains, others free and often massed in clumps. Some of them 
exhibit polar- staining. 

From pus containing all the above forms only the cocci develop 
with any degree of certainty when cultivated on artificial media. 

In broth cultures the threads (4) may be obtained in impure 
culture, but I have only once succeeded in obtaining a pure culture, 
and even then the organisms died out before the proper cultural 
reactions could be ascertained. 

It is obviously impossible therefore to say at present exactly 
which of the above organisms is especially related to the disease, or 
if the various morphological forms cited are only the various phases 
of one or two schizomycetes, or if the various forms are related to 
some higher class of organism. Until careful cultural experiments 
have resulted in a proper determination of these organisms the 
matter cannot be definitely decided. 

Various observers have from time to time investigated the 
bacteria associated with pyorrhoea alveolaris, and of these Galippe 
and Miller deserve notice. 

Galippe (1889) isolated two organisms which produced general 
abscess formation when injected into animals. With one the 
abscesses were frequently met with in the bones and were occasion- 
ally associated with spontaneous fracture, the site of the fracture 
being surrounded with a well defined area of suppuration. The 
second organism produced intermuscular abscesses. These organisms 
were however not properly described, and it is impossible to determine 
anything concerning them. 

Miller conducted a series of cultural and inoculation experiments 
on the subject and came to only negative results. He cultivated 
a number of bacteria upon gelatin and agar from a large series of 
pyorrhoea cases, but was unable to satisfy himself tbat any par- 


ticular organism isolated was the one chiefly concerned in the 
process. He made however several valuable observations, particu- 
larly the infrequency with which the common pus cocci were 
present. Thus in forty-three cases of pyorrhoea examined staphy- 
lococcus aureus was met with three times and staphylococcus albus 

Netter likewise found the pus cocci present in about 10 per cent, 
of the cases examined, and my own cultivation experiments confirm 
those of Miller and Netter, as in one hundred and fifty cases of 
marginal suppuration examined exactly 10 per cent. (15 cases in 
all) gave cultures of the staphylococcus aureus and albus. 

So far my own experiments are very much in a line with 
those of Miller ; I have isolated a large number of different bacteria, 
some of them pathogenic for animals, just as were a number of those 
obtained by Miller, but so far no organism appears with sufficient 
frequency to associate it especially with the disease. The results 
of some inoculation experiments, however, throw some additional 
light upon the subject. Animals (guinea-pigs) succumbed when 
inoculated with the filtrate of old broth cultivations, made from the 
mouth direct, and containing the fine threads referred to above, and 
moreover giving off a considerable faecal smell. No organisms were 
found in the tissues at the _pos£ mortem, and it seems reasonable 
to suppose therefore that the animal died from a toxaemia, especially 
as there were evidences in the haernorrhagic condition of the supra- 
renal capsules that such was the case. 

Such a circumstance appears to point to a toxic element in 
pyorrhoea, and w T e may call to mind the curious shining atrophic 
appearance of the gums in cases of long standing. "What appears 
therefore to be a reasonable supposition is that the particular 
bacteria concerned in the process produce some sort of toxine which 
so alters the vitality of the tissues surrounding the teeth that any 
and every mouth organism may assist in the continuation of the 
process. One of the cultures inoculated was from the mouth of 
a man suffering from various nervous symptoms, including wasting 
and loss of power in the legs, which cleared up on attention to mouth 
hygiene. 1 

Many of the bacteria found in the pus are pathogenic when 
injected into animals. Thus ten guinea-pigs and five rabbits injected 

1 Trans. Odonto. Soc, April, 1902. 


subcutaneously with emulsion of pyorrhoea pus in sterile broth, all 
but one guinea-pig and one rabbit died, in the majority of cases 
within forty-eight hours. 

Some of the animals developed a local abscess at the seat of 
inoculation which when incised contained a thick viscid pus with 
an evil smell. Microscopically this pus contained the same mor- 
phological forms noted in the coverslip preparations from the 
original mouth lesion, but the organisms were not obtained in pure 

The broth cultures showed the same threads of lightly staining 
bacilli and gave off the same unpleasant smell as do tubes inoculated 
from the gum pockets. 

The organisms found at the post-mortem examinations were by 
no means constant, the organism occurring in the largest number 
of cases being a bacillus, generally in jointed chains (streptobacilli). 
This organism was isolated in pure culture from three of the cases 
and re-inoculated into guinea-pigs, each time producing a fatal 
result ; there was no abscess formation, but the organisms were 
recovered from the heart blood in pure culture. 

From two of the cases Staphylococcus albus was obtained, and 
in one case B. mesentericus ruber. Several other organisms were 
also found in various cases, and there is therefore no sufficient 
data to draw any deductions from beyond the general facts that 

(a) the pus is decidedly pathogenic for animals, and that this 
pathogenicity is not apparently due to the common pus organisms ; 

(b) that the organisms growing in broth cultures are able to 
elaborate a toxine (apparently by symbiotic activity) which when 
filtered produces death on inoculation into guinea-pigs. The con- 
dition would therefore appear to be primarily toxic, the suppuration 
with pus formation being a secondary matter. 

The pneumococcus was not met with, nor has Bacillus coli 
appeared on the cultivations except in one case. 

In two cases examined a yeast was obtained which caused death 
when inoculated intraperitoneally into guinea-pigs ; the organisms 
were recovered from the peritoneal cavity in pure culture. Grasset 1 
obtained a pathogenic yeast from a mouth abscess, and the one I 
have isolated appears to be similar. 

Troiser and Achlaime 2 also describe a pathogenic yeast obtained 

1 Arch, de Med. Exp. et Anat. Path. 

2 Fullerton. Joum. of Path, and Bad., 1900. 


from the throat of a patient suffering from enteric fever. This 
organism tended to form hyphae in culture media, and is interest- 
ing in association with the general presence of yeast forms in 
pyorrhoea pus. 

Hunter, 1 in a valuable communication, has called attention to 
the association of various general disturbances related to local 
conditions of oral sepsis, and points out conclusively the relation 
of " septic " gastritis, general septicaemia, and the like, as well as a 
class of toxic conditions of an ill-defined nature, which owe their 
origin to uncleanly and suppurative conditions of the buccal cavity. 

Hunter ascribes the symptoms to "the pus cocci so frequently 
present in the mouth," and quotes Miller, Galippe, Vignal and 
Arkovy. Miller, Netter, and myself, however, agree that the 
common pus organisms are by no means as frequent in the mouth 
as would seem probable — in fact only about 10 per cent, of cases 
examined give cultivations of these organisms. I have already 
shown, however, in the foregoing experiments that the pus of 
pyorrhoea, and the products of the activity of the organisms 
obtained, are extremely pathogenic for animals, thereby confirm- 
ing Hunter's contentions ; and there is no doubt whatever that the 
swallowing of these organisms and their products greatly influences 
the health of certain individuals. All persons with septic mouths, 
however, do not suffer from toxic poisoning, and several of the cases 
from which decidedly pathogenic results followed the injection of 
animals with pyorrhoea pus emulsion exhibited no impairment of 
health in any form, either gastric or general. It therefore becomes 
an interesting problem that toxic absorption does not always 
produce the gastric and other effects noted by Hunter. 

We have already seen in the chapter on immunity that a large 
degree of tolerance may be produced in an animal by repeated injec- 
tions of increasing doses of a given organism or its toxines. I have 
also referred to Ehrlich's theory of antitoxine formation, and we may 
I think apply the conception to an explanation of the tolerance to 
poisons produced in the mouth. 

It is well known that individuals suffering from toxic mouth 
conditions may show no signs of poisoning for long periods, but 
that often such persons develop symptoms of toxaemia somewhat 
suddenly, and that once established the recovery is long and 

' The Practitioner, 1901. 


Eitchie has recently shown that tetanus toxine if mixed with a 
proportion of acid does not give rise to tetanic symptoms when 
injected into a susceptible animal, but a degree of immunity is 
nevertheless produced. 

It is quite possible therefore that some inactivation of the toxine 
produced occurs, and that a certain amount of immunity is produced 
thereby, but that under increased dose the immunity breaks down. 


Bacteria only met with in the Mouth. 

A number of bacteria occur with great regularity in all unclean 
mouths, and wherever any deposit of calculus exists. They ex- 
hibit the peculiarity that they will not grow upon ordinary culture 
media, at any rate when material containing them is inoculated 
directly from the mouth ; they may appear in the abscesses and 
local inflammations produced in animals, by the inoculation of 
material taken from the gum margins. Miller was the first to 
attempt a classification of these mouth organisms, his tabulation 
being as follows : — 

(1) Leptothrix innominata. 

(2) Leptothrix buccalis maxima. 

(3) Bacillus buccalis maximus. 

(4) Spirillum sputugenum. 

(5) Spirochete dentium. 

(6) Iodo coccus vaginatus. 

Miller found that all these organisms refused to grow upon the 
ordinary culture media, and in no case was he able to obtain a pure 
cultivation ; occasionally some of the bacteria grew a little, but they 
soon died out. This question of cultivation is one of the chief 
difficulties in isolating the above organisms, as not only do they 
require special media, but they are particularly susceptible to the 
presence of other bacteria ; the mouth streptococcus, in particular, 
(j rowing down almost all other forms. 

It is probable that the morphological forms tabulated above 
will ultimately prove to be related to more than the six species they 
now represent, but until they have all been obtained in pure culture 
Miller's tabulation should stand. I have succeeded in cultivating 
two at least of the organisms in the above list, and it is to be hoped 
that the others may ultimately be obtained in pure culture. ' Two 
other organisms may be added to Miller's list, one the Leptothrix 


racemosa of Vicentini, and the Streptothrix buccalis described by 

The Group Leptothrix. — A great deal has been written con- 
cerning " Leptothrix," and all sorts of curious things said of the 
doings of the mythical " Leptothrix of tooth decay." Any thread- 
forming organism has been included under the term, so that not a 
little confusion exists in the nomenclature ; more particularly has 
this arisen from the fact that no definite rule has been followed, 
and no proper definition of Leptothrix adhered to. 

Fig. 56. — Showing Vaeious Forms from the Mouth Direct. 

The fine threads are Miller's Leptothrix innominate, the thick chain of bacilli 
B. maximus buccalis. 

The term Leptothrix signifies a genus of bacteria belonging to 
the higher forms of Schizomycetes, and nearly related to the 
Crenothrix and Beggiatoia. 

Zoph, in his classification of bacteria, describes the genus Lepto- 
thrix as " spherical, rod-shaped, and filamentous forms, the last 
showing a difference between the two extremities ; spore formation 
not known, filaments straight or spiral." Migula has proposed as 
a family name for the various higher bacteria, Chlamydobacteriacea3, 
and defines them as " filamentous bacteria composed of rod-shaped 


cells and surrounded with a distinct sheath. Division of the cells 
at right angles to the axis of the filaments. In Phragmidothrix 
and Crenothrix, gonidia are formed by division in three directions 
of space. Eeproduction by gonidia which are motile or non-motile." 
Migula's nomenclature, therefore, is an amplification of the two 
genera of Zoph, Leptotricheae and Cladotricheae. 

The divisions of Migula's Chlarnydobacteriaceae are : — 
I. — Cells without sulphur granules. 
A. — Filaments unbranched. 

(i.) Cell division in one direction of space only (a) (Lcpto- 

(ii.) Cell division before gonidia formation in three direc- 
tions of space. 

(a) Filaments with scarcely discernible sheath 

(Pliragmidiotlirix) . 

(b) Filaments with easily discernible sheath 

B. — Filaments show false branching (Cladothrix). 

II. — Cell contents contain sulphur granules (Thiothrix). 

The Beggiatoaceae are grouped as a separate family. 

Zoph includes Crenothrix, Phragmidothrix, Leptothrix and 
Beggiatoa under the one family Leptotricheae. 

Lehmann and Neumann class Leptothrix, Cladothrix dichotoma 
(F. Cohn), Beggiatoa, Phragmidothrix and Crenothrix under the 
term Higher fusion algae, but disclaim personal knowledge of the 
group. Several species of Cladothrix and Streptothrix are merged 
in the genus Actinomyces. 

From the above brief review of the literature it will be seen 
that considerable diversity of opinion exists as to the proper group- 
ing of these higher bacteria ; Migula's classification appears to be 
the one to which least objection can be taken, and is moreover 
• capable of extension, if necessary, to include new forms. 

Whatever be the exact definition of Leptothrix decided upon, it is 
clear that bacilli which are able at times to form threads cannot 
on that account alone be included as Leptotricheae ; it is, however, 
impossible to state whether the curious bacillary forms often met 
with in the mouth are members of the above family or merely 
involution forms of other bacteria. I am inclined to think that a 
number of the morphological forms often seen are related to the 
Blastomycetes, more particularly as cultivations containing these 


forms often result in the growth of certain yeasts, many of which 
produce distinct filamentous forms on some media, but I am unable 
at present to make any definite statement as to the relationship. 
The yeast filaments often give the granulose reaction. One 
organism that is known as Leptothrix epidermidis, 1 produces coiled 
up and twisted filaments which are slightly motile but show no 
flagella ; the. movement is thought to be due to contractility of the 
organism itself. This organism is said to be common on the skin 
of man. 

It is quite impossible to discuss all the isolated species indi- 
vidually termed " Leptothrix " until fuller knowledge of their 
biology has been obtained ; the species described by various authors 
are therefore given without any attempt at arrangement, but it 
is to be hoped that further work will contribute largely bo our 
knowledge of the subject. 

The organism described by Vignal as Leptothrix buccalis is 
supposed to be identical with Leptothrix buccalis of Eobin. The 
organism described by Vignal is a large bacillus, and is the same I 
have described as B. maximus as far as it is possible to judge from 
Vignal's observations. The organism does not merit the term 
Leptothrix, and Miller's term B. maximus is retained (see below). 

Leptothrix Placoides alba (Dobrzyniecki, Cent. fur. Bdkt., 
B. xxi., 225), obtained only once, from a root filling removed after 
four years. 

Morphology. — Chains of bacilli forming tangled skeins (not 
branching ?) ; on staining, bacillary and cocci-like bodies are seen 
in the centre of the threads. The threads are not motile. 

Staining Beactions. — Stains by Gram's method, and with the 
ordinary aniline dyes, best with aniline-gentian violet or fuchsin. 
Stains blue with acidulated Gram's iodine (granulose reaction). 

Biological Characters. — An aerobic, liquefying, non - motile 
bacillus. Spore formation not observed. 

Gelatin Plates. — In forty-eight hours round minute raised white 
colonies composed of masses of threads resembling the colonies of 
B. anthracis. In three days the gelatin around the colonies becomes 
liquefied and the colony floats in the fluid. 

Gelatin Streak. — Development slow, in four to five days minute 
white colonies appear, liquefaction commencing about the tenth 

1 Byzozero. Cent. fur. Bakt., xx., p. 606., Nos. 16-17. 


A(jar Plates. — Appearance similar to gelatin plate. 

Agar Streak. — A raised clear, cartilaginous mass is formed along 
the streak composed of isolated colonies having the appearance of 
placoid scales. In eight to ten days the colonies have become 
entirely confluent and the whole mass can be removed entire with 
the platinum needle. 

Blood Serum. — Growth similar to agar ; no liquefaction occurs. 

Potato. — No growth. 

Broth. — No growth. 

The organism died out before any more observations were made. 
From the description given of the colonies the organism much 
resembles the Streptothrix group. 


Not yet cultivated and probably including many of the organisms 
of the mouth of thread-forming habit. 

In many mouths, especially those with gingival inflammation, 
with a considerable deposit of "materia alba" around the teeth, 
various thread forms are commonly found. Among these bunches 
of fine slender threads are frequently seen and constitute the 
L. innominata of Miller (see fig. 56). The threads are long (20 n 
and upwards), often curved and twisted, 05 ^ to 0*8 ^ in diameter. 
They rarely show any division into bacillary elements, but occa- 
sionally this may be observed. 

The threads generally show unequal staining when carbol- 
methylene blue is used ; some of these threads stain by Gram's 
methods, others do not. No granulose reaction is given, only a 
yellow colour appearing. 


Obtained by Miller from the deposits around teeth on a special 
medium of " agar-gelatin, dentine glue w T ith 1*5 per cent, of starch 
and sugar." 

No description given beyond the granulose reaction by means, 
of which the organism may be isolated. 

( Yicentini, " Cryptogamic Flora of the Mouth.") 
The LejJtotlirix racemosa of Vicentini was at first credited by its 
discoverer as the origin of all bacteria found in the sputum and saliva,, 

1 Miller, " Micro. Mouth," p. 82. 


morphological form alone being relied upon. Eecently bis views 
have been somewhat modified, but he still considers that many of 
the morphological forms met with in the mouth are phase forms of 
this peculiar organism. The organism in question may be found in 
the majority of mouths, but more plentifully in those mouths where 
little or no care and attention is bestowed upon the teeth. It may 
form thick, whitish deposits upon the surfaces of teeth not opposed 
to one another, and may at times form a thick creamy layer along 
the gum margin, where it is intermingled with other species of 
organisms, many of which have been confused with it. 

Fig. 57. — Leptothrix racemosa of Vicentini, prom Mouth Direct. 
Balsam mount. Photomicrograph and specimen by Dr. Leon Williams. 
x 1,000. 

In the ordinary method of making coverslip preparations from 
the mouth the characteristic form of the organism is very apt 
to be destroyed and the "heads" broken up. Vicentini and 
Williams^ who have worked at this organism, have adopted 
glycerine as a mountant (figs. 58 and 59), by which method the 
various forms are more easily preserved intact. Another method 
which has not been adopted by either of the observers mentioned, 

Dental Cosmos, April, 1899. 


and which gives excellent results, is to examine the organism in 
the hanging drop, saliva or 0-75 per cent, salt solution being used 
as the medium. Such preparations are of course not permanent. 

The organism, according to Vicentini and Leon Williams, 
belongs to a higher order than the Bacteria or Schizomycetes, and 
it is suggested that it should be placed among the Fungi. Williams 
thinks that the process of sporulation that he has seen is nearly 
allied to the Uridineae or Busts. In coverslip preparations, and 
particularly in the hanging drop made from the white deposit con- 

Fig. 58. — Leptothrix racemosa of Vicentini, from Mouth Direct, 
showing " Fruitful Heads." 

Glycerine mount. Photomicrograph and specimen by Dr. Leon Williams. 
x 1,000. 

taining this organism, curious felted masses of entwined threads are 
seen, many of which appear as if surrounded with closely adhering 
cocci. Some of these masses are finger-shaped and project from 
the general mass of the threads and cocci (see fig. 59). After a little 
search isolated specimens can be found, when the cocci-like bodies 
are seen to be arranged in regular order and, according to Williams 
and Vicentini, attached to the thread by basidia which, according to 
the former, are seen to pass from the thread to the " spore." The 
•central thread can easily be traced through the mass of spore- like 


bodies to its free end, the whole appearance reminding one of the 
common "Friar's Cowl" of the hedge-rows when ripe. Williams. 
gives a number of photographs of this organism, some of which 
show the cocci-like bodies particularly well, but it is by no means 
certain that these basidia are not artifact. If the "spores" are 
attached with these basidia or short stalk-like processes of the- 
central thread, one would expect to observe the basidia on free 
spores, or on parts of the thread, which so far has not been 
accomplished. If. on the other hand, the spores are attached one- 
to another they are more referable to the type of some of the 
moulds (cf. Penicillium). 

| g Fig. 59. — Fruitful Heads of L. racemosa, showing Arrangement 


Photomicrograph and specimen by Dr. Leon Williams, x 2,000. 

The threads of this peculiar organism have been shown by- 
Williams to stain in a special way with a modification of the Gram- 
method, as follows : — the material containing the threads, &c, is 
carefully made into an emulsion with distilled water and then 
stained with hot aniline gentian violet for from eight to ten minutes. 
The specimen is then placed in hot iodine solution to fix, washed 
in absolute alcohol and then counterstained with methylene blue ;. 


by this method of staining, spore-like bodies are seen to occupy the 
ends of the segments of the threads. The spore-like areas may be 
stained in another way, which rather precludes their description as 
spores. A coverslip preparation is made and MacConkey's capsule 
stain ' poured on. The preparation is then warmed until steam 
commences to be given off (it must not be allowed to boil), the stain 
is left on the coverslip for five minutes, washed off and the cover- 
slip mounted. The curious beaded appearance of the threads is 

Fig. 60. — Leptothrix Racemosa, Mouth Direct. Stained Capsule Stain. 
Showing darkly staining dots. x 1()00. 

brought out by this method; the stained " spores " are more to be 
regarded as arthrospores rather than true endogenous spores. On 
staining with Miller's iodine or with iodine acidulated with sulphuric 
acid, some of the areas apparently corresponding to the areas that 
stain with the foregoing method take a faint blue or violet tinge — 
in other words, they give the granulose reaction. 

Good specimens of this organism are difficult to obtain, and 
great care must be exercised in making the coverslip preparation. 

1 Dahlia, -5 gm., methyl green (00 crystals), 1-5 gm., sat. alcoholic fuchsin, 
10 cc, water to 200 cc. 


The best method to adopt is to suspend some of the material 
containing the organism in distilled water. A large drop is trans- 
ferred to a coverslip and allowed to dry — anything like spreading 
should be avoided. Flaming the coverslip is also liable to break up 
the organism, and it is best to fix with alcohol and ether as in 
staining blood-films. 

When Vicentini first sent the description of the organism to 
Miller the latter was of the opinion that it should be classified as 
a Cladothrix or Crenothrix rather than a Leptothrix, but upon the 
representation of Vicentini he withdrew his objection. Williams, 
while accepting the term Leptothrix provisionally, has shown that 
the " fruitful heads" may be not inaptly compared to the fructifica- 
tion of the Cordiceps militarius and Botritis Bassini, 1 providing 
the sterigmata and basidia exist. At the same time the method of 
sporulation or fructification of the Cladothrix and Crenothrix have 
some points in common with the organism under discussion. 

The gonidia or asexual spores of Botritis Bassini are supported 
upon well-marked sterigmata or basidia, the term basidium being 
used in both a general sense when it is applied to the end of the 
thread that undergoes asexual sporulation, and in a special sense, 
when it is used to indicate the stalk upon which the asexual spore 
or gonidium is carried and by which it is attached to the parent 

In Penicillium the carpophore or special spore-bearing hypha is 
an erect branch of the mycelium, the terminal portion of which 
divides into numerous branchlets which in turn divide up into a 
chain of naked gonidia without special sterigmata, a condition with 
a little modification that is not unlike the sporulation of the L. 

Again, the Crenothrix breaks up into a multitude of spore-like 
bodies, the terminal portion of the thread undergoing multipartate 
division into gonidia ; these may be extruded or remain attached to 
the interior of the thread. It is also to be noted that if a freely 
growing coccus, such as Staphylococcus albus, be grown in the 
presence of an equally freely growing bacillus, coverslip preparations 
made from the mixed culture will show cocci apparently attached 
to the thread forms and to the shorter bacilli as well, much in the 
same way as the preparation from the mouth shows cocci attached 

1 Du Bary, " Morphology of Fungi," p 65. 


threads supposed to be the early sporulation of the L. racemosa. 
It is, moreover, not difficult to produce the appearance of sterigmata 
by using a stain like gentian violet, which is notorious for its quick 

There are many other points of similarity between the L. 
racemosa and some of the Ascomycetes, for further particulars of 
which the reader is referred to Du Bary's book. 

As the organism we have been discussing has as yet defied 
attempts at cultivation, it is difficult to assign it to any particular 
genus. Its morphology does not so far conform with any one 
class of Ascomycetes or Schizomycetes. 

Fig. 61. — Bacillus Maximus. 
From agar cultivation twenty-four hours old at 37 "5° C. Stained Gram. 
x 1,000. 


Leptothrix buccalis (Vignal). Bacillus maximus, Leptothrix 
buccalis maxima (Miller). 

The largest of the mouth bacteria occurs in most dirty mouths 
and is often seen associated with the " Leptothrix " innominata. 

Miller, who first applied the term B. maximus, describes another 


organism, Leptothrix buccalis maxima, which differs in two par- 
ticulars only from B. maximus — the threads do not give the granulose 
reaction and the segments are of greater length. Neither of these 
organisms were obtained in pure culture, and the differentiation is 
therefore not sufficient to class them as of different species. 

Vignal's Leptothrix buccalis is a bacillus, and in its cultural 
characters corresponds closely to those of the large bacillus I have 
obtained in pure culture from the mouth (see fig. 61). Under these 
circumstances Miller's term Bacillus maximus is adopted. 

Under favourable circumstances this organism may be obtained 
in pure culture by making an emulsion in broth of the material 
containing the bacillus and other mouth bacteria ; the emulsion is 
then plated on a series of (1) maltose agar, or (2) potato gelatin 
plates and the colonies carefully examined for the organism sought 
for. The bacillus requires some time before it becomes acclimatised 
to the conditions of artificial media, but when it has developed its 
" laboratory habit " it grows freely. 

Although the organism forms endogenous spores I have never 
obtained cultivations from the mouth direct by the method of 
differential sterilization (see p. 19). 

Morphology. — Thick jointed threads 0'5 to 1*5 p- broad, 10 to 
20 /* long ; some threads may be much longer, and occasionally 
twisted, especially upon old potato cultures. The individual ele- 
ments are 1 to 4 v long but may be much longer. 

From the mouth direct and in the cultures the bacilli show 
considerable irregularity of the protoplasm which is brought out 
on staining. The " spotted " appearance of the bacilli when stained 
by carbol fuchsin or carbol methylene blue is due to a fragmentation 
of the cytoplasm. Staining by hot carbolic fuchsin shows spores 
situated at the ends of the threads, while large, clear unstained 
areas may be met with which appear to be spaces left by the 
extruded spores, and also to the effects of plasmolysis. 

Staining 'Reactions. — Stains by Gram's method, and by the 
ordinary aniline dyes. With carbol-methylene blue red granules 
often appear in the older threads. With MacConkey's capsule stain 
isolated masses of deep staining occur in the interior of the threads. 
With Moller's method well marked dark red spores are found. 
By staining with Pitfield's method a few lateral flagella are to be 
found ; they are not present in great numbers, but may be as many 
as six lateral and one terminal. On the threads only a few lateral 


flagella on isolated segments are found. A few of the threads give 
the granulose reaction. Large oval involution forms appear in old 

Biological Characters. — An aerobic, facultative anaerobic, liquefy- 
ing motile bacillus. Forms spores. 

Gelatin Plates, 22° C. — At the end of twelve hours white, grey, 
flat, round colonies with entire edge. The central area is darker. 

Microscopically, f obj. — Edge irregularly dentate, yellow ; centre 
dark brown, irregular, the whole finely fibrillated (marmorated). 

Gelatin Stab, 22° C. — Liquefaction in twenty-four hours with 
cone-shaped depression (infundibuliform). Growth occurs along 
the line of stab below the liquefaction. The apex of the cone is 
occupied by a thick flocculent precipitate. A slight flocculent scum 
appears upon the surface, and numerous flocculi appear in the 

Gelatin Streak, 22° C. — Well marked groove of liquefaction 
generally in twenty-four hours. The fluid is filled with flocculi. 

Agar Plates, 37 -5° C. — Brownish, round, raised colonies, similar 
to gelatin plate. 

Agar Streak. — Brownish-grey streak with irregular, flocculent 
edge. The surface is distinctly granular, especially when seen 
through a small lens, and has the appearance of frosted glass. It 
does not become corrugated. The film is easily removed with the 
spatula, and does not become dry as does B. subtilis. The bacilli 
form long articulated threads with well marked spores. 

Blood Serum, 37*5° C. — Twelve hours: raised, moist, grey, edge 
much less irregular than on agar. No liquefaction occurs. 

Potato, 37*5° C. — Twenty-four hours : well marked, grey, moist 
growth limited to the inoculated area, and showing no tendency to 
spread. Later the growth becomes a dirty grey. 

Broth, 37*5° C. — Twelve hours : flocculi white to dirty grey, 
which fall and produce a thick flocculent precipitate ; very little 
general turbidity. A flocculent pellicle may form at a later stage. 
The bacilli are motile, but not excessively so. No indol reaction. 

Litmus Milk. — Well marked acid reaction, no clot is formed. 

Glucose, Lactose, Maltose Broth. — Well marked acid reaction in 
two days. No gas is given off. 

No gas formed on gelatin shakes. Grows aniierobically on 
glucose formate media, but not so well as aerobically. No gas 
on glucose formate broth. 


Pathogenesis. — Undetermined. 

Since the first description of this organism in 1898 I have 
obtained similar organisms from twelve cases. 


Considerable interest is attached to the fact that comma- shaped 
and spiral bacteria are present in a large number of mouths, and 
that in certain conditions of pathological importance, particularly 
ulcerative stomatitis, vibrios are present in great quantities, so much 
so, in fact, that Bernheim 1 considers them the active agents. They 
are always present during subacute inflammatory conditions of the 
mucous membrane of the mouth ; are frequently met with asso- 
ciated with B. diphtherise upon the tonsil — in fact in several cases 
I found them alone with the Klebs-Loefner bacillus. They are also 
found in the cavities of tubercular lung (Artrault), 2 and are not 
uncommonly found in antral suppuration, where I have met with 
them in seven cases. Spirilla, having the same characteristics in 
that they are excessively difficult to obtain in pure culture, are 
frequently present in faeces, and in one case of Bright's disease 
spirilla were present in the peritoneal fluid. 

Miller, in 1883, first called attention to the presence of the 
comma - shaped bacilli in the mouth, and a year later Lewis 
described organisms resembling the cholera vibrio in the oral 
secretions of healthy persons. 

The researches of Miller carried on for two or three years with 
a very large variety of culture media did not result in the isolation 
of a pure cultivation of these spirilla. Colonies did occasionally 
develop, but never withstood the transference to a second tube of 
media. At the same time Miller obtained cultivations of several 
species of spirilla, two of which he discusses. 

The one proved to be the Vibrio Finkler-Prior, the other was a 
non-motile organism producing curious twisted and contorted forms, 
some resembling the capital letter O. The old cultures formed 
streptococci like involution forms. It appears evident from Miller's 
work, and from my own experience, that the spiral and comma- 
shaped bacteria of the mouth do not all belong to the same species ; 
although in all ''dirty " mouths {i.e., with deposits of calculus or 

1 Semaine Medicate, 1896. 

2 Arch, de Parasitologic, Tome i., No. 2, 



Fig. 62.— Spirillum Sputugenum (Spirilla). 

From mouth direct ; gentian violet stain, x l.oCO. 

Fig. 63. — Spieillum Sputugenum (Comma forms). 
From mouth direct ; gentian violet stain, x 1,000. 


" materia alba "), spiral or comma- shaped forms are constantly 
found. These spirilla are extremely difficult to obtain in pure 
culture, and it was only after two years of experiment, during which 
forty different species of media were tried, that a culture was 
obtained. In many cases the organisms grew for a short time, but 
died as soon as they were transferred to another medium. 

The mouth spirillum generally grows for a short time upon blood 
serum, and may also be observed upon several fluid media but only in 
small numbers. Thus on beer wort, mucin broth (made from snails), 
saliva filtered and 1 per cent, peptone added, maltose broth with 
0-05 per cent, of potassium sulphocyanide, egg broth and alkali 
albumin broth, all will occasionally show a limited development. 
The two media which I have found to give the best results are 
saliva set with agar, and potato gelatin ; the labter medium is not 
a favourable one for the mouth streptococcus, which otherwise 
grows to the exclusion of most other forms. 

The method of obtaining the spirilla is as follows : successive 
streaks are made upon a number of tubes of the potato medium, 
and in three or four days a second series of tubes is streaked with 
any minute pin's point colonies which show spirilla. The second 
series may often require to be treated as the first, and even then 
the organisms have a great tendency to die out. 

"When a culture is obtained it requires subculturing every few 
days for two or three weeks, after which it gradually becomes accus- 
tomed to the altered conditions and develops fairly well. The early 
cultures do not form typical spirilla ; under the hanging drop, however, 
the characteristic movements are seen. 


Morphology. — Vibrio, occurring in young cultures as comma- 
shaped rods 0*1 to 0*3 ^ in breadth, 1 to 2*5 /u. long, with rounded 
or pointed ends. In old cultures well marked spirilla are formed, 
some composed of commas united in series, others of spirilla with 
three or four turns without a break. Very long threads are also 
met with ; these are often thinner and irregular (spirochete). 
Spiral forms best marked on both cultures in forty-eight hours. 
No endogenous spores were found, but irregular spherical bodies 
are found attached to the older threads (see fig. 66) as well as 
independently (? arthrospores). 

Staining Reactions. — Does not stain by Gram's method, best 


with carbol fuchsin, or carbol gentian violet, or aniline gentian 
violet. The organism only stains faintly with carbol methylene blue. 
In old cultures the threads stain unequally, and give the appear- 
ance of chains of bacilli with unstained intervals. With Pitfield's 
method single terminal flagella are seen. 

Biological Characters. — An aerobic facultative anaerobic, lique- 
fying motile spirillum. Does not form spores (endogenous). 
Arthrospores formed (?). 

Gelatin Plates, 22° C. — At the end of forty-eight hours minute 
greyish-white colonies, much like streptococci, appear, they are 
moist and flat, and the gelatin around them soon commences to 

Fig. 64. — Spirillum sputugenum, freshly Isolated from the Mouth. 
(Spirillum form not yet well developed.) x 1,000. 

Microscopically, § obj. — Brownish, round, or oval, not granular, 
with darker opaque irregular centre. 

Gelatin Stab, 22° C. — Cup-shaped liquefaction (napiform), in 
four days, little fluid ; the tube may often be inverted without any 
of the contents escaping ; white flocculi appear in the fluid and a 
considerable deposit at the bottom. 

Gelatin Streak, 22° C. — Groove of liquefaction in three days 
with white flocculi in fluid. No pigment is produced. 


Agar Plates, 37-5° C— Brownish, flat, smooth, moist, central 
portion elevated, edge entire or gyrate. 

Agar Streak, 37*5° C. — Good growth in twenty-four hours with 
defined, entire edge slightly raised, grey, translucent. Later the 
growth becomes buff coloured. 

Blood Serum, 37-5° C. — Grey, smooth, moist streak. The 
medium is slowly liquefied. 

Fig. 65. — Spirillum sputugenum. 
Agar cultivation at twenty-four hours (comma forms). Stained gentian 
violet, x 1,000. 

Litmus Milk, 37*5° C. — Well marked acid reaction in twenty- 
four hours, with coagulation of casein in five days. Not 

Broth, 37-5° C. — In twenty-four hours general turbidity with 
slight pellicle. A four days' culture gives a well marked cholera- 
red reaction (nitroso-indol), with nitrite free sulphuric acid; H 2 S 

Potato. — No apparent growth in twenty-four hours at 37*5° C. ; 
two to three days at 22° C. well marked rich red-brown colouration, 
moist and shiny. Involution forms and threads common. 


Glucose, Lactose, Maltose Broth. — Well marked acid production 
in forty-eight hours ; gas evolved. 

An'derobiosis. — Grows on glucose formate media without oxygen 
in Buchner tubes, and produces gas on glucose formate broth. 

Pathogenesis. — Pathogenic for guinea-pigs (four only inoculated), 
1 cc. of agar culture emulsion fatal in three days when injected into 
peritoneal cavity. 

Fig. GG.— Spirillum sputugenum. 
Broth cultivation at seven days, showing involution forms ( ?arthrospores). 
X 1,000. 


Probably identical with foregoing. Occurs in fine irregular 
threads, 0*1 /* wide, 5 to 7 /* long. Sometimes seen with coccus- 
like bodies attached to thread (? arthrospores). Found in deposits 
along gum margins. Stains with difficulty with carbol methylene 
blue and not by Gram's method ; so far I have not been successful 
in observing flagella. 

The twists or turns of the organism are more angular than those 
of the ordinary spirillum, and the motility appears confined to quiet 
revolution upon their long axis. 



A good deal of interest has been awakened with regard to this 
group since the diphtheria and tubercle bacilli were shown by Hueppe 
and his pupil Fischel, as well as the earlier work of Eoux, Nocard, 
and Metchnikoff, to occasionally exhibit distinctly branched forms. 

Still later Nocard, Eppinger, Moeller, and many others have 
shown that some branching fungi of the actinomyces class are 
" acid-fast," that is to say, they resist decolourization with weak 
(25 per cent.) solutions of mineral acids when stained with hot 
carbolic fuchsin, just as do the tubercle, leprosy and smegma 
bacilli. Portions of the threads are under these circumstances 
almost indistinguishable from the tubercle bacillus, especially when 
the examination has to be made in sputum, as cited in the case 
described recently by Birt and Leishman. 1 

Mycologists differ somewhat as to the proper nomenclature of 
the group of fungi of which the " ray fungus " or " actinomyces " is 
the type. The majority of English and American pathologists 
adopt the term Streptothrix for the group comprising the organisms 
which have been variously termed, Actinomyces Cladothrix, 
Oospora, and Nocardia. Lehmann and Neumann class the genus 
as Actinomyces. 

For the present at any rate Streptothrix is perhaps the better 
term, and is adopted here. The characteristics of the genus are 
briefly : — delicate threaded organisms free of chlorophyl, showing a 
true branching mycelium; portions of the threads show club-shaped 
endings and gonidia which may also be developed in the mycelium. 
Fragmentation of the mycelium occurs with the production of 
various morphological forms not to be differentiated from the various 
forms of Schizomycetes. The mycelium may be developed from the 
gonidia or from the fragmented threads. 

S. actinomyces has already been described (p. 124). 

A short time since I described (Trans. Odont. Soc, June 18, 1899) 
an organism isolated from the mouth .which resembled the Cladothrix 
dichotoma of Cohn, as far as I was then able to judge, from the 
Cladothrix dichotoma in the Guy's Laboratory. The organism 
in question is, however, to be placed in the same category as the 
actinomyces bovis, and conforms to the generic description of 
Streptothrix given above ; it should therefore be termed Streptothrix 

1 Journal of Hygiene, Ap., 1902, p. 120. 


Since the first description of that organism was published I have 
met with it upon many occasions in the mouth, particularly in the 
thick viscid pus sometimes emanating from the gum pockets 
around the teeth affected by pyorrhoea alveolaris. On one occasion 
it appeared in the heart blood of a rabbit dying from the injection 
of pyorrhoea pus. The organism obtained from this source conforms 
to the general characteristics of the other specimens obtained by 
cultivation. I have not met with it, so far, in normal mouths. 
There is therefore some reason to suppose that it may have a 
relationship to alveolar pyorrhoea. 


Obtained from pyorrhoea pus, and from the white deposits 
around the teeth, also in gingival inflammation. 

Morphology. — Filamentous forms ; in young cultures a tangled 
mycelium is produced, the threads of which show well marked 
lateral branches ; the ends of those threads are frequently club- 
shaped. Dichotomous branching or branching by longitudinal 
fission of terminal filaments not observed. The lateral branches are 
unequally distributed upon the thread and often present a constric- 
tion at their junction with the main stem. The terminal portions 
of the threads may taper to a point or show distinct enlargement 
(fig. 67). 

Somewhat later the threads and clubbed extremities undergo 
changes resulting in the thread staining unequally, especially with 
gentian violet or Gram's method ; at the same time segmentation 
transverse to the long axis results in the production of bodies which 
may be termed gonidia. Fragmentation now occurs, the mycelium 
splitting up into a series of forms morphologically simulating 
various bacteria (fig. 69), and as some of the threads are slightly 
spiral, comma shapes and spirilla are also formed. 

The gonidia set free germinate and produce threads by lateral 
extension, and thus the cycle is complete. On solid media parti- 
cularly the colonies become covered with a white powder consisting 
of the gonidia which, if transferred to another tube, grow into 

Staining Reactions. — Stains by Gram's method and by the 
ordinary aniline dyes. The clubs take the stain most deeply. Not 
acid-fast to carbol fuchsin and 25 per cent, sulphuric acid. 



Fig. 67. — Streptothrix buccalis from Mouth Direct. 
Stained Gram, showing branched threads and clubs, x 3,000. 

Fig. C8.— Streptothrix Buccalis. 
Forty-eight hours' cultivation on agar stained Gram. Showing branched 
threads, x 600. 


Biological Characters. — An aerobic, liquefying streptothrix. 

Gelatin Plates, 22° C. — At the end of three to four days (rarely 
earlier) minute, hard, raised, colourless and spherical colonies 
appear, which increase somewhat rapidly and occasionally form 
cone-shaped projecting points the tops of which become coated with 
a white powder. Liquefaction commences somewhat later and the 
colonies gradually sink into the fluid gelatin. Occasionally the 
colonies do not produce the projecting cone-shaped points but 
remain flat and ring-shaped, the centre remaining clear. 

Fig. 69. — Streptothrix buccalis. 
Seven days' cultivation on potato stained Gram, showing involution forms 
due to fragmentation of the threads, x 1,000. 

Gelatin Stab, 22° C. — Slight development occurs along the line of 
inoculation in minute beaded colonies. Liquefaction commences at 
the surface about the fifth day and extends to the tube walls ; the 
liquefied medium is separated from the solid by a horizontal plane 

Gelatin Shake, 22° C. — No gas bubbles are produced, and a faint 
cloud of minute colonies, best marked at the surface, appears in four 
to five days. Liquefaction stratiform. 


Fig. 70. — Streptothrix buccalis. 
Five days' cultivation in agar. 


Agar Plates, 37-5° C. — In twenty-four hours small, hard, convex 
colonies, having a cartilaginous consistency ; in three or four days 
they become truncated cones, and later surmounted with white 
powder composed of gonidia. The cones often crack across the 
summit. The colonies eventually produce a slight depression in the 
agar ; they are extremely difficult to remove with the platinum 
needle, and to make a satisfactory coverslip preparation it is best to 
crush a colony between two cover glasses. 

Blood Scrum, 37'5° C. — No discolouration on this or any other 
media, with the exception of an occasional colony in freshly isolated 
specimens. Liquefaction occurs. 

Litmus Milk, 37 -5° C. — In two days no change, later precipita- 
tion of casein occurs, the milk clearing and taking on a bluish- 
purple tinge. Morphologically, mostly streptobacilli. 

Broth, 37*5° C. — Flaky particulate scum gradually developing 
into spherical colonies which fall to the bottom. A layer is generally 
retained around the meniscus, which becomes chalky white. 

Potato, 37-5° C. — Yellowish or orange-brown discolouration, not 
constant, with colonies flat or raised and about the size of small 
shot. A chalky-white appearance is later caused by the formation 
of gonidia. 

Spore Formation. — No endogenous spores observed ; cultures 
killed by exposure to 75° C. for ten minutes. Motility not present. 
No sulphur grains seen. 

All the cultures give off a characteristic musty smell like a damp 



Saprophytic Bacteria of the Mouth not Described in 
Previous Sections. 


Found extremely widely distributed, in water, in faeces, in 
certain diseases of animals and in diseases of men. Commonly pre- 
sent in milk, &c. Often present in the mouth. Frequently present 
in abscesses situated near digestive tract. 

Morphology. — Bacilli 1 to 4 M long, and 04 to 0*6 a* broad. Often 
forms rods of considerable length. The ends are rounded. Actively 
motile in young cultures. 

Staining Beactions. — Stains easily with the usual methods, but 
not by Gram's method. In old cultures polar staining occurs. The 
fiagella may be stained by Pitfield's or van Ermengem's methods ; 
they are generally twelve, rarely more than fourteen, arranged 
around the bacilli (peritrichic). 

Biological Characters. — An aerobic, facultative anaerobic, non- 
liquefying motile bacillus. No spore formation. Grows well at 
37° C. and at the ordinary room temperature (22° C.). 

Gelatin Blates, 22° C. Superficial Colonies. — At first small 
yellowish punctiform colonies, later becoming large irregular ; edge 
lobulate or dentate, shining. Centre opaque and white and often 
a little raised. 

Deep Colonies. — Punctiform, later wheatstone-shaped, yellowish. 
No liquefaction. 

Microscopically. — Irregularly marked surface, showing faint, 
irregular stripes or traversed by vein-like markings as in marble 

Gelatin Stab, 22° C. — Well marked growth to bottom of stab, 
whitish-grey, granular ; no liquefaction. Surface thin, whitish- 
green, flat, edge notched. 


Gelatin Shake, 22° C. — Three days: well marked cloud of gas 
bubbles ; no liquefaction. 

Gelatin Streak, 22° C. — Spreading, white, thin, granular, as on 
surface of stab. 

Agar Streak, 37*5° C. — Grey-white, glistening, moist, translucent. 
Condensation water clear with slight precipitate. 

Potato, 22° C. — Yellow to yellow-brown discolouration of potato 
occasionally occurs. 

Fig. 71. — Bacillus coli commune. 
Agar cultivation twenty- four hours old. x 1,C00. 

Litmus Milk, 37'5° C. — Coagulation and marked acid reaction. 
H 2 S and indol produced. 

Broth, 37-5° C. — Dense turbidity with thick sediment. Indol in 
seven days or less. 

Lactose, Maltose, Glucose Broth. — Acid fermentation and gas 
given off (C0 2 ). 

Anaerobiosis. — Grows well on glucose formate media, producing 
much gas on glucose formate broth. 

Pathogenesis. — Variable, some cultures producing death in one to 
five days, with general septicaemia when injected intraperitoneally, 


Subcutaneous injection may produce local necrosis of skin. Appears 
in blood stream a few hours before death in some diseases, and 
often present in the blood three or four hours after death. 

(Dobrzyniecki, Cent, fur Bah., Bd. xxi., p. 835.) 

Found in dental caries. 

Morphology. — Bacilli 1*5 m long, irregular in size, non-motile. 
Stains by Gram's method. 

Gelatin Plates. — Minute punctiform colonies in two days; later 
the colonies become larger, round, well denned, and golden-yellow 
in colour. 

Gelatin Stab. — Development occurs to the bottom of the stab 
and is a light yellow colour below and golden-yellow above. The 
gelatin is not liquefied. 

Agar Streak. — In forty-eight hours a golden-yellow moist 
streak is formed, darker in the centre than the edge. 

Potato. — Similar to agar ; no colouration of the medium occurs. 

Pathogenesis. — A slight local reaction produced in mice and 
guinea-pigs, but no general reaction. 


Synonym, — Bacillus g, Vignal. 

Found by Vignal in the salivary secretions of healthy persons. 

Morphology . — A very short bacillus, with round ends, almost as 
broad as long ; in cultures upon agar the length is from 0*5 to 1 m, 
usually about 0*7 h- ; in neutral bouillon it is from 1 to 1*7 h- long. 
In old cultures involution forms are common ; in stained prepara- 
tions the two ends are more deeply stained than the central portion. 

Biological Characters. — An aerobic, liquefying, chromogenic 
bacillus. Produces a yellow pigment. Spore formation not 
observed. Motility not mentioned. Grows slowly at the room 

Gelatin Plates. — At the end of forty-eight hours the colonies are 
round, with refractive contour and of a mastic-yellow colour ; they 
are but slightly elevated, and the gelatin commences to liquefy 
around them. 

Gelatin Stick Cultures. — At the end of forty-eight hours a yellow- 
ish-white growth is seen along the line of puncture, and upon the 
surface a layer having the same colour and several millimetres in 


diameter has developed ; by the fourth day the surface growth has 
increased to twice the size and is yellow at the centre, while the 
periphery is white. The growth along the line of puncture is 
abundant, and consists of small, closely crowded colonies ; below 
the surface growth a cup-shaped cavity filled with clouded liquefied 
gelatin is seen. By the sixth day a small funnel of liquefaction 
has formed, the liquefied gelatin is clear, and contains some white 
flocculi in suspension. By the twelfth day the gelatin in the tube 
is completely liquefied, an abundant yellow deposit is seen at the 
bottom, and the liquefied gelatin has the same colour. 

Surface of Agar. — Golden-yellow plaques are developed, which 
are easily removed with the platinum needle. 

Bouillon. — A thin, iridescent pellicle is formed upon the surface, 
and the fluid below is clouded, while an abundant yellow deposit 
accumulates at the bottom. Does not grow well in acid bouillon. 

Potato. — At the end of forty-eight hours a thin and extended 
layer is formed of a yellow colour, which later has a brownish tint. 


Synonym. — Bacillus j, Vignal. 

Found by Vignal in the salivary secretions of healthy persons. 

Morphology. — Bacilli with square ends, from 1-4 to 3 m long; 
often united in pairs, the elements of which may be joined at an 
augle of greater or less degree. 

Biological Characters. — An aerobic, liquefying bacillus. Spore 
formation not observed. Motility not mentioned. Grows at the 
room temperature in the usual culture media. 

Gelatin Plates. — At tbe end of forty-eight hours, small round 
colonies are developed, which increase considerably in thickness and 
diameter, and by the fourth or fifth day have caused liquefaction 
of tbe surrounding gelatin. 

Gelatin Stick Cultures. — At the end of forty-eight hours a small 
mass has formed at the point of inoculation, and a scanty line of 
development is seen along the track of the inoculation needle ; 
by the fourth day the growth has extended over the entire surface 
and presents a decided prominence at the centre. The gelatin 
below is liquefied, and remains transparent with some opaque white 
flocculi in suspension ; by the twelfth day the liquefaction extends 
to a depth of 2 cm., and a yellowish-white, abundant deposit is 
seen at the bottom. 


Surface of Agar at 36° to 38° C. — Small, white, opaque colonies 
are developed which present a small, nipple-like projection at the 

Bouillon. — A diffuse cloudiness is produced ; a thick, dull white 
layer forms upon the surface, and an abundant dull white deposit is 
seen at the bottom of the tube. Does not grow well in acid bouillon. 

Potato. — A rather thick growth is developed, which extends 
slowly and acquires a slightly pinkish tint. 


Obtained by Vignal in cultures from healthy buccal secretions. 

Morphology. — Bacilli with square ends, straight or slightly 
curved, about 0*5 /* in diameter and varying greatly in length, 
from 1*5 to 6*5 ^ ; often united in chains. 

Biological Characters. — An aerobic, liquefying bacillus. Spore 
formation not observed. Motility not mentioned. Grows rather 
slowly at the room temperature, more abundantly at 37° C. 

Gelatin Plates. — At the end of twenty-four hours, at a room 
temperature of 18° to 20° C, prominent, small, grayish-white 
colonies are developed upon the surface. At the end of forty-eight 
hours a collarette with irregular festooned margins is developed 
around this central mass ; this is thinner and much more trans- 
parent than the central portion of the colony. Under a low power 
it is seen to be formed of an innumerable series of skein-like 
bundles, arranged side by side and more or less twisted, which 
proceed from the central mass. 

Gelatin Stick Cultures. — At the end of forty-eight hours, a small, 
flat mass is developed at the point of puncture, and a scanty growth 
is seen along the line of inoculation. 

On the fourth day the superficial growth covers the entire 
surface, it is translucent by transmitted light, and white by reflected 
light. By the sixth day the gelatin is liquefied to a depth of 
1 cm. below the superficial growth ; the liquefied gelatin remains 
transparent ; upon the surface is seen a white, membranous 
layer, and at the bottom a rather scanty white deposit ; liquefaction 
slowly extends downwards, and by the twelfth day has reached a 
level corresponding with the bottom of the line of puncture. 

Surface of Agar. — At the end of twenty-four hours at 36° to 38° 
C., a dull white layer, having a thickness of about 1 mm., is 
developed ; this is easily broken up with the platinum needle. 


Bouillon. — A slight cloudiness is quickly produced, a thin film 
forms upon the surface, and a scanty white precipitate at the bottom 
of the tube. 

Potato. — At the end of forty-eight hours a layer the size of a 
five-franc piece is developed, which has a pale pink colour and a 
rough surface — resembliDg a lichen. 

Blood Serum. — Is liquefied rather rapidly, and acquires a 
brownish colour, while an abundant white precipitate accumulates 
at the bottom of the tube. 


Found by Vignal in the salivary secretions of healthy persons. 

Morphology . — Bacilli with slightly rounded ends from 0*8 to 
1*2 ijl in length when cultivated upon agar, and from 1*4= to 2*4 M 
when cultivated in neutral bouillon ; usually solitary, occasionally 
united in short chains. 

Biological Characters. — An aerobic, liquefying bacillus. Spore 
formation not observed. Motility not mentioned. Grows rather 
slowly at the room temperature — more rapidly at 37 -5° C. 

Gelatin Plates. — At the end of forty-eight hours, small, projecting, 
opaque, white colonies are developed ; at the end of four days the 
colonies are seen as conical, opaque, white masses divided into 
about twenty segments by grooves which start from the summit. 

Gelatin Stick Cultures. — A small but prominent white mass is 
seen at the point of puncture, and a scanty line of development along 
the track of the inoculating needle ; on the fourth day the surface 
growth has extended nearly to the walls of the tube, and just below 
this some fine branches are given off from the line of the growth ; 
the sixth day the entire surface is covered, and the gelatin below is 
liquefied for a short distance ; the eighth day the liquefaction has 
extended downward, and the solid gelatin below has a clouded 
appearance owing to the development of a quantity of small white 
colonies ; by the twelfth day the liquefied gelatin has a depth of 
about 2 cm., a shining, white mycoderma is seen upon the surface, 
a white deposit at the bottom, and below this numerous small 
colonies in the solid gelatin. 

Surface of Agar. — Very adherent, white colonies are formed, 
which later extend to form a transparent white membrane. 

Bouillon. — A slight cloudiness is produced, and a very scanty, 
whitish deposit is seen at the bottom of the tube. Does not 
develop well in acid bouillon. 



Blood Serum. — A whitish layer is formed, which later becomes 
semi-transparent, and causes a slow liquefaction of the medium. 

Potato. — At the end of forty-eight hours a layer is developed 
which has a velvety appearance in the centre, and a yellowish or 
brownish-white colour ; by the end of forty-eight hours this layer 
is as large as a five-franc piece. 

Fig. 72. —Vibrio Finklee-Peioe. 
From twenty-four-hours-old culture, x 1,000. 

(42) VIBRIO FINKLER-PRIOR (V. Proteus). 

Occasionally found in the mouth (Miller). Occurs in certain 
diarrhoea attacks. 

Morphology. — Curved and bent rods 2-4 a* long, 0-4 to 0*6 p broad ; 
forms commas and spiral threads. 

Staining Beactions. — Not by Gram, best with dilute carbol 
fuchsin or aniline gentian violet, and clearing in absolute alcohol. 

Gelatin Plates, 22° C. — Eound, edge entire, yellowish, granular. 
Microscopically, § obj., yellow, centre darker, edge coarsely granular. 
Liquefaction in twenty-four hours. Occasionally the colonies have 
hair-like projections (ciliate). 

Gelatin Stab, 22° C. — Saccate (sleeve shaped) liquefaction pro- 
gressing very rapidly ; the fluid remains turbid and a well marked 
pellicle is formed. 

Agar Plate, 37"5° C. — Colonies similar to gelatin. 

Agar Streak, 37*5 C. — Moist, yellowish rather; slimy and 

Blood Serum, 37-5° C. — Moist, regular, well marked groove of 


Potato, 22° C. — Well marked yellow, slimy, shining layer. 

Litmus Milk, 37*5° C. — Coagulation of casein, which is re- 
dissolved ; slight acid reaction. 

Broth, 37*5° C. — General turbidity and pellicle formed. Indol 
reaction slight or absent. 

Glucose Formate Broth, 37*5° C. — Grows aniierobically, but no 
gas produced. 


(Diplococcus roseus, Flugge.) 

Widely distributed organism, very common in air, frequently 
present in mouth. 

Morphology. — Round, oval and irregular cocci (0*6 to 1*0 /* in 
diameter), often occurring in masses or in pairs. 

Staining Reactions, — Stains well with the ordinary aniline dyes 
and by Gram's method. 

Biological Characters. — An aerobic, chromogenic coccus ; gelatin 
slowly liquefied. Not motile (? Micrococcus agilis of Cohn). Grows 
best at 22° on ordinary media, also at 37*5° C. Pigment only formed 
in presence of air. 

Gelatin Plates, 22° C. — Irregular, round or crenated, raised, 
small, rose-red colonies on surface, the deep colonies not developing 
much. The colonies gradually sink into the gelatin. Under f obj., 
round or lenticular, entire edge, finely granular and pale rose-red in 

Gelatin Stab, 22° C. — Fine thread-like growth along stab, gelatin 
very slowly liquefied. Surface lobed and irregular, rose-red. 

Gelatin Shake, 22° C. — Growth of colonies only near surface, 
little in depths. No gas. 

Agar Streak, 37*5° C. — Smooth, shining, regular edge in twenty- 
four hours. Condensation water clear, and later, with red precipitate. 
Colour best developed at 20° C. 

Potato, 22° C. — Glistening, rose-red, and often with outer white 
zone, often raised and lobular ; medium not coloured. 

Litmus Milk, 37*5° C— No change. 

Broth, 37*5° C. — Slight turbidity, with rose-red precipitate, 

On potato cultures of Micrococcus roseus the colonies are a much 
brighter red. 


A number of other cocci producing a red pigment have been 
described, but they are all apparently related to the Micrococcus 
roseus. Micrococcus lactericeus (Freund, Cent, fur Bakt., Bd. xxi., 
834) differs slightly, but is probably a variety. Bacillus roseus 
{Trans. Odont. Soc.,. June, 1898) is probably the same; the bacilli 
were very small and much resembled cocci. 

The Sarcina roseus is thought by Lehmann and Neumann to be 
a "form" of the Micrococcus roseus (for further particulars see 
Lehmann and Neumann, p. 192). 



The Microscope. 

For bacteriological work a good compound microscope is neces- 
sary aud should be fitted with the following : — 

Objectives. — f, £, and T V oil immersion. 

Substage condenser. — Abbe or other pattern. 

Nose piece. 

Coarse and fine adjustment. 

Mechanical stage. 

The microscope consists of several parts which will be considered 

Fig. 73. — Compound Bacteriological Microscope. 

The Stand. — The pattern of stand is not of great importance, but 
the " tripod" is the most convenient for general work. It is most 
important that the stand be rigid and should allow of the body being 
tilted as far as the horizontal position in stable equilibrium. 

The Stage. — Two species of stage are in use : (a) the plain, 
(b) the mechanical ; and for bacteriological work the latter is 


preferable, but not absolutely necessary. Those mechanical stages 
which are fixed by screws to the ordinary plain stage rarely work 
well for any length of time without developing a "kick," thereby 
throwing the object out of focus whenever the stage is adjusted. 
In selecting a stage care should be taken to observe that the move- 
ment in both directions is free from kick, and moreover sufficient to 
allow of plate cultivations, &c, being examined. This refers also to 
the plain stage. 

The Substage Condenser. — Various forms of condenser are in use. 
The Abbe consists of a plano-convex and a concavo-convex lens and 
is the one generally in use. By means of the condenser the light is 
focussed upon the object, otherwise stained preparations cannot be 
brought sharply into focus when the ^ obj. is in use. The condenser 
should be fitted with an iris diaphragm to regulate the light. 

The Mirror. — Should have both plane and convex surfaces. The 
plane surface is to be used with the condenser. 

Body Tube. — This tube carries the objectives and the eye-piece. 
The continental microscopes have a short body tube, the British a 
long tube, and the objectives are severally adapted. The body tube 
should be capable of extension, but it is essential to have a rack 
and pinion adjustment. 

Focusing Adjustments. — The coarse adjustment is essential for 
all bacteriological work ; .by its action the objective is lowered till 
almost in focus and the focusing then completed with the fine 
adjustment. There are several forms of fine adjustments, and one 
should be chosen which does not carry more than half the weight 
of the body tube (see fig. 74). 

The Objectives. — The § obj. should give a perfectly flat field and 
sharp definition. The ^ oil immersion lens should give good Central 
definition with no blurring ; the edges of the field must be free from 
colour refraction. The lens should also allow of the diaphragm 
to be opened to its full extent without causing any blurring of the 

Great care is necessary in selecting lenses as it is impossible to 
make all lenses of uniform standard. Diatoms are not entirely 
satisfactory in testing a lens ; blood films stained with eosin and 
bacteria stained with fuchsin give much better tests. 

The oil should always be wiped off the -^ after use with a clean 
piece of wash-leather kept for the purpose. If any dirt has been 
allowed to collect on the field lens it is best to clean it off with some 
immersion oil and the leather. 



Only gross carelessness will account for canada balsam upon the 
y 1 ^, and very great care must be exercised in removing it, otherwise 
the lens cement may be dissolved and the objective ruined. 

Eye Pieces. — A low and high power eye-piece are convenient, 
the former magnifying about 5 diameters, the latter about 12. 

Fig. 74. — Fine Adjustment of Microscope. 


(1) Unstained Specimens, Hanging Drop Slides, dx. — Use the J 
first always ; it generally gives sufficient magnification for the pur- 
pose. Back the lens down with the coarse adjustment until it 
almost, but not quite, touches the coverslip, then while looking 
through the microscope rack upwards until the object comes into 
view, then proceed with the fine adjustment. When using the 
condenser the flat side of the mirror should be employed, and with 
the ^ obj. the condenser requires lowering. 

(2) Stained Specimens. — Use the T V obj., taking care that no 
canada balsam is on the surface of the coverslip. Place a drop 
of immersion oil (cedar wood) in the middle of the coverslip, and 
with the coarse adjustment lower the lens till it touches the oil 
and all but touches the glass, then looking through the microscope 


carefully rack up with the coarse adjustment until the object comes 
into view, finally focus with the fine adjustment. The diaphragm 
should be well open and the substage condenser raised to its limit. 

Source of Light. — Daylight is the best, but — at any rate in London 
— is so uncertain that it is best to accustom oneself to a constant 
source of light. An argand burner or an incandescent mantle give 
the best results. It is an excellent plan to use a condenser in the 
form of a spherical flask, filled with water tinted with a neutral 
blue to correct the yellowness of the gas light ; the water also 
removes the greater part of the heat rays. When not in use the 
microscope is advantageously kept covered with a glass bell-jar to 
exclude dust. 

Hanging drop specimens should be made of all organisms 
examined. In examining the specimens under the microscope, 
rack down the condenser and use the ^ obj. and close the iris 
diaphragm. All specimens of living bacteria require to be 
examined with the diaphragm nearly closed. With the stained 
specimens, on the other hand, rack the condenser close up to the 
slide and open the diaphragm when looking for the bacteria ; with 
tissue preparations the diaphragm requires closing to bring out the. 
tissue structure. 


Coverslips. — For smear preparations the coverslips must be 
entirely free from grease, &c, otherwise good specimens cannot be 

The new slips are cleaned as follows : — 

(1) Boil for thirty minutes in a strong solution of chromic acid. 

(2) Wash with distilled water until no more yellow colour is. 
seen in the washing water. 

(3) Einse in rectified spirit three times to remove the water. 

(4) Wash in absolute alcohol twice. 

(5) Transfer to a glass jar of absolute alcohol, using a pair of 
clean forceps which are kept for coverslips alone. 

The coverslips must not be touched with the fingers when in 

Old coverslips, such as hanging drop preparations and slides, 
when finished with should be placed in 2 per cent, solution of lysol. 
The balsam becomes converted into soap and the slips are easily 
removed. They are then boiled in strong soap solution (Hudson's 



Extract or Sapon) and then cleaned, as the new ones, in chromic 
acid, &c. The glass slips may be also boiled in soap solution and 
wiped dry with a clean cloth. The use of strong alkali spoils the 

Glass apparatus, beakers, Erlenmeyer flasks, &c, should be 
washed out with stroDg soap solution and a flask brush, rinsed and 
drained. Agar and gelatin if allowed to dry in the flasks is very 
difficult to remove. 


(From Chester's "Determinative Bacteriology.") 

Gelatin Stab Cultures. 
A. Non-liquefying: — 
Line of puncture. 

Filiform. — Uniform growth with special characters (fig. 75, i.). 
Nodose. — Consisting of closely aggregated colonies. 
Beaded. — Consisting of loosely placed or disjointed colonies 
(fig. 75, ii.). 

-"O r~n po <^n 


Fig. 75.— Character of Gelatin Stab Cultures. 
i., Filiform; ii., beaded; iii., tuberculate-acinulate ; iv., villous; v., arbor- 
escent. (Eyre, after Chester.) 

Papillate. — Beset with papillate extensions. 
Echinate. — Beset with acicular extensions (fig. 75, iii.). 
Villous. — Beset with short, undivided, hair-like extensions 
(fig. 75, iv.). 



Plumose. — Delicate feathery growth. 
- Arborescent. — Branched or tree-like, beset with branched hair- 
like extensions (fig. 75, v.). 
B. Liquefying : — 

Crateriform. — Saucer- shaped liquefaction of gelatin (fig. 76, i.). 
Saccate. — Shaped like an elongated sac, tubular, cylindrical 

(fig. 76, ii.). 
Infundibuliform. — Shaped like a funnel, conical (fig. 76, iii.). 
Napiform. — Shaped like a turnip (fig. 76, iv.). 



£>4 r^> 


Fig. 76. — Chaeactees of Liquefaction in Gelatin Stab Cultuees. 
i., Crateriform; ii., saccate; iii., infundibuliform; iv., napiform; v., fusi- 
form; vi., stratiform. (Eyre, after Chester.) 

Fusiform. — Outline of a parsnip, narrow either end, broadest 

below the surface (fig. 76, v.). 
Stratiform. — Liquefaction extending to wall of tube and then 
downward horizontally (fig. 76, vi.). 
Plate Cultures. 
A. Form. 

Punctiform. — Dimensions too slight for naked eye determina- 
tion, minute, raised, semispherical. 
Bound. — Of more or less circular outline. 


Fusiform. — Spindle shaped, tapering either end. 
Cochleate. — Spiral and twisted like a snail shell (fig. 77, L). 
Amoeboid. — Very irregular streaming (Proteus) (fig. 77, ii.). 
Mycelioid. — Filamentous, with the radiate character of a 

mould (fig. 77, hi.). 
Filamentous. — An irregular mass of loosely interwoven 

filaments (fig. 78, i.). 
Floccose. — A dense woolly structure. 

Fig. 77. — Plate Cultures: Types of Colonies (Form). 
i., Cochleate; ii., amoeboid; iii., mycelioid. (Eyre, after Chester.) 

Fig. 78.— Types of Colonies : Plate Cultures (Form). 
i. , Filamentous ; ii., rhizoid ; iii., conglomerate; iv., toruloid. (Eyre, after 

Rhizoid. — Of an irregular branched, root-like character (fig. 

78, ii.). 
Conglomerate. — An aggregation of colonies of similar size 

and form (fig. 78, iii.). 
Toruloid. — An aggregation of colonies like a budding yeast 

plant (fig. 78, iv.). 
Rosulate. — Shaped like a rosette. 
B. Surface Elevation. 

(1) General character as a whole. 



Flat. — Thin leafy spreading over surface (fig. 79, i.). 
Effused. — Spread over surface as a thin, veilly layer, more 

delicate than the preceding. 
Baised. — Growth thick, with abrupt terraced edges (fig. 79, ii.). 
Convex. — Surface the segment of a circle but very flat (fig. 

79, hi.). 
Pulvinate. — Surface the segment of a circle but decidedly 

convex (fig. 79, iv.). 
Capitate. — Surface hemispherical (fig. 79, v.). 

Fig. 79. — Characters of Surface Elevation of Plate Cultures, Non- 
Liquefying Stab Cultures. 
i., Flat; ii., raised; iii., convex; iv., pulvinate; v., capitate; vi., urnbili- 
cate; vii., umbonate. (Eyre, after Chester.) 


Fig. 80.— Structure of Colonies (Microscopic). 
, Clouded; ii., moruloid ; iii., grumose in centre. (Eyre, after Chester.) 

(2) Detailed characters of surface. 

Smooth. — Surface even without any of the following dis- 
tinctive characters. 
Alveolate. — Marked by depressions separated by thin walls 

resembling a honeycomb (fig. 81, ii.). 
Punctate. — Dotted with punctures like pin-pricks. 
Bullate. — Like a blistered surface, rising in convex promin- 
ences, rather coarse. 
Vesicular. — Covered with minute bubbles or vesicles due to 
gas, much finer than bullate. 


Verrucose, — Wart-like, bearing wart-like prominences. 

Squamous. — Scaly, covered with scales. 

Echinatc. — Beset with pointed prominences. 

Papillate. — Beset with nipple-like projections. 

Bugose. — Short irregular folds, due to shrinkage of surface 

Corrugated. — In long folds due to shrinkage. 
Contoured. — An irregular but smoothly undulating surface, 

like the surface of a relief map. 
Rimose. — Abounding in chinks, clefts or cracks. 
G. Internal Structure of Colony (microscopic). 

(1) Refraction weak. — Weak outline, and surface of relief 
not well defined. 

Fig. 81.— Structure of Colonies (Microscopic). 
i., Reticulate; ii., alveolate; iii., gyrosej iv. , marmorated. (Eyre, after 

(2) Refraction strong. — Outline and surface of relief well 
denned, dense not filamentous 
Amorphous. — Without definite structure as specified below. 
Hyaline. — Clear and colourless. 

Homogeneous. — Uniform structure throughout colony. 
Homochromous. — Colony uniform throughout. 
Finely Granular. 
Coarsely Granular. 
Grumose. — Coarser than preceding, particles in clustered 

grains (fig. 80, iii.). 
Moruloid. — Segmented into more or less regular segments 

(fig. 80, ii.). 
Clouded. — A pale ground with ill-defined patches of deeper 
colour (fig. 80, i.). 


Reticulate. — In the form of network, like the veins of a leaf 

_ (fig- 81, i.)- 
Areolate. — Divided into rather irregular, angular spaces by 

more or less definite boundaries. 
Marmorated. — Showing faint, irregular stripes, or traversed 
by vein-like markings as in marble (fig. 81, 
Gyrose. — Marked by lines like the rivers of a map (fig. 81, iii.). 
Rimose. — Showing chinks, cracks, or clefts. 

Filamentous colonies. 
Filamentous. — As already defined. 

v. vi. vii. vm. ix. 

Fig. 82.— Characters of Edges of Colonies. 
i., Entire; ii., undulate; iii., repand ; iv., erose ; v., lobate-lobulate ; vi. 
auriculate; vii., lacerate; viii., fimbriate; ix., ciliate. (Eyre, after Chester.) 

Floccose. — Filaments closely packed. 

Curled. — Filaments in parallel strands like locks of hair or 
D. Edges of Colonies. 

Entire. — Without toothing or division (fig. 82, i.). 

Undulate. — Wavy (fig. 82, ii.). 

Repand. — Like the border of an open umbrella (fig. 82, iii.). 

Erose. — As if gnawed, irregularly toothed (fig. 82, iv.). 

Lobate. — Blunt, rounded projection of edge (fig. 82, v.). 

Lobulate. — Minutely lobate. 

Auriculate. — With ear-like lobes (fig. 82, vi.). 

Lacerate. — Irregularly cleft as if torn (fig. 82, vii.). 

Fimbriate. — Fringed (fig. 82, viii.). 

Ciliate. — Hair-like radial extensions (fig. 82, ix.). 



Optical Characters : — 

Transparent. — Transmitting light. 

Vitreous. — Transparent and colourless. 

Oleaginous. — Transparent and yellow, olive to linseed-oil 

Resinous. — Transparent and brown varnish or resin coloured. 
Translucent. —Faintly transparent. 
Porcelainous. — Translucent and white. 
Opalescent. — Translucent, greyish white by reflected light, 

smoky brown by transmitted light. 
Nacreous. — Translucent, greyish white with pearly lustre. 
Sebaceous. — Translucent, yellowish to greyish white. 
Butyrous. — Translucent and yellow. 
Ceraceous. — Opaque and wax coloured. 
Cretaceous. — Opaque and chalky white. 

Dull. — Without lustre. 
Glistening . — Shining. 


Indol. — Inoculate glucose free broth tubes with organism to be 
tested, incubate at 37*5° and 22° C. for ten days. 

Test : Add ten drops of pure (nitrite free) concentrated sulphuric 
acid to each broth tube and 1 cc. of a 0*03 per cent, solution of 
sodium nitrite. 

A pink colouration in ten minutes at room temperature indicates 

Plicnol. — Place 50 cc. of the broth culture to be tested in a flask 
connected with a condenser, add 5 cc. pure concentrated hydro- 
chloric acid. The distillate is collected and divided into three 
portions : — 

(a) Add a few drops of Millon's reagent and boil ; a red colour 
indicates phenol. 

(b) Add a few drops of bromine water ; turbidity if phenol is 



(c) Add a few drops of dilute ferric chloride ; a violet colour 
indicates phenol. 

Reduction of Nitrates to Nitrites. 
Inoculate medium composed of : — 

Peptone .. ... ... ... ... ... ... 10 gm. 

Sod. nitrate 0*02 gm. 

Water 1,000 cc. 

Test the water first and also use blank control tubes. 
To the cultivation add a mixture of equal parts of the following 
solutions. — 

I. — Naphthylamine 1-0 gm. 

Water 100 cc. 

II.— Sulphanilic acid 1*5 gm. 

Dilute acetic acid ... ... ... ... ... 150 cc. 

A pink coloration denotes reduction of nitrates to nitrites ; the 
control tube often shows a pink colour, but if any change has 
occurred in the culture tube the colour is distinctly deeper. 

Ammonia. — To 100 cc. of culture add 2gm. of calcined magnesia, 
and distil. The distillate gives a yellow colour with Nessler's 
reagent, the tint of which is proportionate to the amount, and may 
be estimated in the usual way. 

A control sample of uninoculated broth should also be distilled 
Nesslerised, and the tints compared. 

Sulphuretted Hydrogen. — May be tested for by using broth 
or gelatin containing iron saccharate or tartrate ; or a piece of 
lead acetate paper may be suspended in the culture tube. 

Diastatic Ferment. — Inoculate sugar free broth, and after several 
days' incubation mix the culture with thin starch paste containing 
2 per cent, thymol. Place the mixture in the incubator for eight 
to ten hours, filter and test filtrate with Fehling's solution. 

For separation of acids see Chester's " Determinative Bac- 
teriology," p. 39. 

b^ g 5 

fe .2 


3 S 3 2 
6q C ^ H 


c3 c3 

w % 

'5 *" 

CI Pi« 


« fe hh 02 02 ^ 
O O 

3 W 

o y el 

if ° 

° d -3 & p Q 

3 <! a O g 3 

• e3 _2 -S ,-3 ,£3 

3 g ~2 02 CQ Ph 02 

fi | J H O0 02 .2 

D O 

a oq 

-g ^ 3 j 3 g" 





Abscess, alveolar 

. 170 

Acid and dental caries . . 

. 139 

„ production 

. 22 

Active immunity 

. 72 

Agar, ordinary nutrient 

. 55 

Age and sex relation to caries 

. 137 

Agglutination . . 

. 77 

, , method of testing . . 

. 76 

Alkali albumin 

. 109 

,, production 

. 22 

Alveolar abscess 

. 171 

,, ,, bacteria in 

. 172 

,, pyorrhoea 

. 175 

Ammonia, test for 

. 226 

Anaerobic bacteria 

. 17 

,, caries 

. 17 

„ cultivations 

. 62 


. 16 

Aniline water 

. 45 

Anthrax immunity and B. pyocyaneus . 

. 123 

Anti-bacterial bodies . . 

. 73 

,, sera 

. 76 


. 72 

Anti-pneumococcic serum 

. 101 


37, 39 

,, action on staphylococci 

. 95 

,, choice of 

. 37 

,, Lockwood's solutions of 

. 38 

,, tests of strength of . . 

. 38 

Antitoxic bodies 

. 72 

,, sera.. 

. 75 


. 75 

,, estimation of 

. 75 

Aphthous stomatitis 

. 174 

Arkovy on dental caries 

. 145 

Artificial caries 

. 133 

,, immunity 

. 72 

,, ,, production of 

. 73 




Attenuation by air 



,, antiseptics 



,, heat 




Avine tuberculosis 

.. 112 

Bacilli allied to the diphtheria bacillus . . 





B. (Vignal) .. 

.. 210 


buccalis fortuitus 

.. 209 


,, rninutis 

.. 208 


coli commune 



,, ,, in tooth pulp 

.. 170 


dentalis viridens 

.. 130 





fluorescens liquefaciens 



„ non-liquefaciens 






G. (Vignal) . . 



gangramae in tooth pulps 



,, pulpse 

.. 129 








.. 208 





,, biological characters . . 

.. 193 


,, staining reactions 



mesentericus fuscus 

129, 156 


, , in tooth pulps 

.. 16S 


,, ruber 



,, vulgatus 







.. 165 


pulpae pyogenes 




. . 121 





salivarius sepiicus (Biondi) 

.. 92 





tuberculosis . . 


Bacteria and moisture 



,, temperature 

.. 19 


chemistry of cell 



classification of 



effect of light on 






in coal 



in dental caries 



in dento-alveolar abscesses 



in dust 



in pyorrhoea alveolaris 




Bacteria in the air 
. ,, in tooth pulps 

,, only met with in the mouth 

,, rate of development 

,, structure of . . 
Bacterial film in caries . . 

,, plaques, experimental production of 
Bactericidal power of saliva 

Baumgarten's classification 
Black on lime salts in teeth 
Blastomycetes in alveolar abscesses 
Blood, collection of 
,, films 

,, serum, coagulated 
,, „ inspissation 

Blue pus 

Boston's spring forceps 
Branched forms of diphtheria bacillus 
Broth nutrient 
Brownian movement 
Buchner's tubes 
Bullock's apparatus for anaerobes 

Cane sugar 

Capsule of pneumococcus 

„ staining 
Caries, anaerobic 

,, and diet 

,, and flour 

,, and lime salts 

,, artificial 

,, bacteria of 

,, bacterial plaques 

,, dental . . 

Changes associated with immunity 
Characters of bacterial cultures.. 
Chart for study of bacteria 
Chemical reactions of cultures . . 
Chemiotaxis, negative . . 

„ positive 

Chemistry of bacterial cells 

„ of food stuffs 

Chester's nomenclature of cultural characters 
Choice of antiseptics 


Choquet on dental caries 

,, inhibition of 

Chronic suppurative parotitis 

,, buccalis 

Classification, Chester's 

,, Lehman and Neumann 

,, of bacteria general 

Cleaning apparatus 

,, slides, lysol 

Coccus salivarius septicus 
Colour production of B. gang. pulp. 
Cornet's spring forceps. 
Coverglass films 

,, preparations 

Coverslip cleaning 

,, jar .. 
Culture media. . 

„ ,, for mouth spirilla 

Cultures, shake 

,, stab . . 

,, streak 
Czenzynke's stain 

Decalcification by trichloracetic acid 

Dental caries 

Dentine, caries of 


Diastatic ferment, test for 

Diet and dental caries . . 

Differential sterilization 

Diphtheria bacillus 

,, allied species 

„ biology 

,, diagnosis of 

,, in healthy mouths 

,, in milk 

,, in urban districts 

,, in water 

,, morphology 

,, Neisser's stain 

,, occurrence 

persistence in throat 
resistance to drying 
toxine formation 



Diphtheria bacillus, varieties of. . 



Diplococcus pneumoniae 


Diseases associated with streptococcus 


Disinfection of the hands 

Dobrzyniecki on dental caries . . 

Embedding tissues 

Enzymes in dental caries 

,, inter-cellular 

,, intra-cellular. . 

, , separation of 

,, tests for liquefying 
Epidemic parotitis 
Erlenmeyer flask 
Erlich's theory 
Estimation of anti-toxine 
,, of toxine .. 
Exaltation of virulence 
Examination of cultures 
Experimental caries and bacterial plaques 

Facultative anaerobes 

Fats .. 

Fermentation of carbohydrates . . 

,, ,, lactic acid 

,, ,, wine must 

Films, coverglass 
„ from blood 
,, from liquid media 
Filtration of toxine 
Fixation of coverslip films 

,, of tissue preparations 
Flagella stains 
Fluorescens, group of bacilli 
Food stuff chemistry 
Food supply of bacteria 
Forceps, Boston's 

,, Cornet's 
Fragmentation of streptothrix buccalis threads 
Frankel's pneumococcus 

Galippe on pyorrhoea 

Gas-forming bacteria in alveolar abscesses 

,, formation 

Gases, action of 


Gelatin nutrient 
Genus leptothrix 

,, streptothrix 
Glands, tubercular, and septic teeth 
Glass apparatus, sterilization of 

Goulard's fixative solution 
Gram's method of staining 


Hands, disinfection of . . 
Hanging drop preparation 

,, drop slides 
Hay, bacillus 
Hearson's incubator 
Heat produced by anaerobes 

,, production 
Higher bacteria 
Hoffmann's bacillus 
Hot air sterilization 
,, water filtration 
Hunter on septic mouths 

Immune serum 


changes associated wi 

to influenza bacillus 
to pneurnococcus 
Immunization, active 

,, artificial 

Incubator, Hearson's 
Indifferent gases 
Indol, test for . . 
Influenza bacillus 

„ immunity to 
, , morphology 
,, pathogenesis 
,, staining reactions 
Inhibition of chromogenesis 

,, ,, growth 
Inoculation of animals.. 
,, ,, culture tubes 
,, ,, liquid media 
,, ,, solid media 
,, ,, wires 



Intra-cellular enzymes 

Intermittent sterilization 

Interstitial nephritis caused by staphylococci 

Instruments, sterilization of 

Inter-cellular enzymes 

Iodine Grams 

Iodococcus magnus 

Irregularities of tests and caries 

Kirk, on alveolar abscess 
Koch's tuberculin 


Leber and Rottenstein's experiments 
Lehman and Neumann on Pluorescens group 

,, ,, ,, on Micrococcus pyogenes 

Leptothrix classification 

, , group 

,, innominata.. 

,, Migula on . . 

,, placoides alba 

,, racemosa 

,, ,, allied species 

,, ,, sporulation 

,, ,, staining reactions 

„ Zoph on 

Light effect on bacteria 
Lingelsheim on streptococci 
Liquefaction of dentine matrix . . 
Litmus neutral, for colouring media 
Lockwood's antiseptic solutions. . 
Loemer's blood serum 
Lophotrichic arrangement of flagella 
Lower bacteria 
Lysogenic action 
Lysol for cleaning slides 

MacConkey's capsule stain 
MacFadyen on the tubercle bacillus 
Malay carbohydrate fermentation 

Marmorek on streptococcus 
Martin, Sidney, diphtheria toxine 
McCrorie's flagella stain 
Media, beer wort gelatin 

,, blood agar 

,, blood serum 

,, bread 

• cultivation 




Media, gelatin agar 

glucose formate agar 
glycerine agar . . 
,, broth 

,, gelatin 

inosit-free broth 
iron salt 
litmus milk 

neutralization of litmus method 

nutrient agar . . 
„ broth . . 
,, gelatin 
peptone water . . 
phenolphthalein method 
potato . . 

,, gelatin ,. 
sugar agar 
„ peptone water 
Mesentericus group 

,, group in tooth pulps 


Metchnikoff on immunity 
Method of isolating mouth streptococci): 

,, of testing agglutination 
Micrococcus gingivae pyogene^ 
pyogenes . . 

, , morphology 

,, pathogenesis 


Microscopical defects in caries . . 
Miller on bacteria in tooth pulps 
,, on pyorrhoea alveolaris .. 
,, on spirillum 
Milk sugar 

Moisture and bacteria 
MOller's method for spores 
Monkeys' mouth bacteria 

,, mouths and putrefaction 
Monotrichic bacilli 
Morphology of diphtheria bacillus 
,, ,, pneumococcus 
,, ,, staphylococcus .. 
,, ,, streptococcus 



Morphology of streptothrix 

,, ,, tubercle bacillus . . 

,, variations of . , 
Morse, staphylococcal nephritis 
Motility of bacteria 
Mouth streptococcus 

„ vibrios.. 
Mycosis of tonsil 

Native races and caries 

Natural immunity 

Neisser's stain 

Neutralization, litmus method . . 

,, phenolphthalein method. . 

Nitrites, test for 
Notes on the use of the microscope 

Organisms in dental caries 

Parasites, facultative 

,, obligatory 
Parotitis, epidemic 

,, suppurative 
Passage, method of 
Passive immunity 
Pasteur-Chamberland niters 
Pasteur theory of exhaustion 
Pathogenesis . . 

,, of mouth streptococcus 

,, of staphylococcus 

Pathogenic bacteria of the mouth 
, , effect of pyorrhoea pus 

Pathology of infection with diphtheria bacillus 
Peptone water 
Peptonization of gelatin 
Periostitis, aveolar dental 
Peritrichic flagellation . . 
Persistence of diphtheria bacilli in throat 
Petri dishes 

Petruschky on streptococci 
Pfeiffer's reaction 
Phenol, test for 

Pigment, alteration of by media 
Pigments of B. pyocyaneus 
Pitfield's nagella stain . . 


Plate cultures . . 

biology of 
morphology of 

,, thermal death point 


,, growth ou gelatin 

,, in pneumonia . . 

,, in saliva 

,, in tooth pulps . . 

,, morphology 

,, pathogenesis 

,, staining reaction 

Porcelain filters 

,, ,, sterilization of . . 

Potato bacilli . . 

,, cutter . . . . . . 

,, media 
,, tubes . . 
,, ,, Roux 
Preparation of antitoxine 
Principles of staining 
Production of immunity 
Products of putrefaction 
Proteus vulgaris 
Pseudo diphtheria bacillus 
Pseudomonas pyocyanea 

Pulp, dental, bacteriology of 
Pure cultures 
Pyocyaneus, bacillus 

,, ,, cultural characters 

,, ,, pathogenesis 

,, ,, pigments .. 

,, ,, varieties of 

Pyorrhoea alveolaris 

,, ,, bacteria in . . 

,, ,, effect of filtered cultures 

,, „ Galippe on . . 

Rack for Petri dishes 

Rate of development of bacteria . . 

Ray fungus 

Reaction of medium 

Read on roller flour 



Resistance of spores 

Retention theory of immunity . . 

Robins' leptothrix 

Roux on diphtheria bacillus 

Roux's potato tubes 

Saliva, bactericidal power 

,, collection of 
Sanarelli, experiments with saliva 
Saprophytic bacteria 
, , of mouth 


,, aurantiaca 
,, lutea .. 
Sections of teeth to show organisms in situ 
Separation of enzymes . . 

,, of toxines 

Septic gastritis 
Serum, antibacterial . . . . . . 

,, antitoxic 
Side chains, Erlich's 
Sidney Martin on diphtheria toxines 
Sloped media 
Spirillum sputugenum . . 

,, „ cultural characters 

,, ,, in diphtheria 

,, ,, in pyorrhoea alveolaris 

,, ,, in stomatitis 


,, dentium 

Spore, determination of presence of by heat 
,, formation 
,, germination 
,, resistance of 

, , aniline gentian violet 

,, ,, water 

„ capsule stains . . 

,, carbol-fuchsin .. 

,, ,, methylene blue .. 

,, ,, thionin blue 

,, contrast for Gram 

, , for spores 

„ general principles 

,, Gram's method. . 

,, Gram, Muir and Ritchie. . 

,, ,, Weigert . . 

, , Loemer's methylene blue 


Stains, Neisser's for B. diphtheria 

,, Ziehl-Neelsen for acid-fast bacteria 
Standardization of media 
Staphylococcal nephritis 
Staphylococcus albus . . 

,, in the mouth 
,, inhibition of liquefaction 
aureus . . 

., antiseptics, action of 

,, biology 

from dead teeth 
in alveolar abscess 
,, in endocarditis 

in pyorrhoea alveolaris 
,, morphology 
,, occurrence 
„ pathogenesis 
,, staining reactions. . 

salivarius pyogenes, Biondi 

Steam sterilizer 

Sterilization at low temperature 
by filtration 
by hot air. . 

by steam under pressure 
by streaming steam 
necessity of 
of glass apparatus . 
of instruments 
of porcelain filters . 
Sterilized swabs 
Sternberg on antiseptic testing- 
Streak cultivations 

,, ,, from pyorrhoea . 


Streptococcus, biology of 
brevis . . 
isolation of 
morphology of 
of mouth 

septo pycemicus, Biondi 
staining reactions 

.. 147 

.. 96 


.. 94 



. . 170 

.. 96 

.. 177 

.. 94 

.. 93 


.. 94 

.. 98 

.. 92 

147, 172 

. . 143 






.. 85 

87, 150 

86, 90 

. . 82 

.. 92 

. . 84 



Streptococcus, varieties of 


biology . . 


granules in pus 

method of infection 


Structure of bacteria . . 
Study of cultivations . . 
Sugar fermentation 

,, media .. 
Sulphuretted hydrogen, test for 
Suppurative parotitis . . 

,, of B. tetanus and pyocyaneus 

Temperature and bacteria 

Test media 

The Microscope 

Theories of immunity 

Theory of retention 

Thermal death point 

Tissue preparations 

Tomes on lime salt in teeth 

Toxic effect of pyorrhoea cultivations 
„ symptoms in pyorrhoea alveolaris 

Toxine of diphtheria bacillus 

Toxines, action of 

,, estimation of . . 

,, filtration of 

,, production of, by bacteria 

,, separation of . . 

Trichloracetic acid for decalcification 

Tubercle bacillus 

,, action of antiseptics 
,, ,, of dead 

,, ,, of light on 

„ and septic teeth 
,, biological characters 
,, carious dentine 
,, immunization to 

,, staining reactions 
,, thermal death point 
„ tissue reactions 

Tuberculin, preparation of 

Typhoid fever vaccination 


Ulcerative stomatitis 

Yan Ermengem's flagella stain . . 
Variations in virulence of B. diphtherias 
Varieties of B. diphtheria} 
of B. pyocyaneus 
„ of streptococcus 
Vibrio Finkler-Prior 

,, of the mouth 
Vigual's anaerobic method 

,, leptothrix 
Virulence, exaltation of 

,, of pneumococcus 

Wallace, Sim, on dental caries . . 
Washbourn's anti-pneumococcic serum 

,, blood agar 

Widal's reaction 
Wright's method 

Yeasts, pathogenic 
Yersin's diphtheria toxine 


. . 173 

.. 110 

. . 104 
.. 123 

.. 84 
194, -21-2 


.. 194 

.. 184 

91, 99 

.. 134 
.. 101 
.. 98 



Ziehl Neelsen's stain 




























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